具有肿瘤多药耐药逆转作用的金雀异黄素衍生物的合成及生物活性研究

细胞膜P-糖蛋白(P-gp)介导的药物外流是肿瘤多药耐药(MDR)产生的重要机制,异黄酮类化合物可以通过抑制P-gp活性发挥MDR逆转作用.通过对P-gp抑制剂进行结构分析,以金雀异黄素为母体,在其7位、8位及4'位分别引进碱性边链,设计、合成了20个金雀异黄素衍生物(其中16个未见文献报道),并检测了其多药耐药逆转活性.结果表明,大多数目标化合物对人白血病耐药细胞株K562/A02具有不同程度的耐药逆转作用.其中目标化合物8a,8b,8d,8e逆转作用较强,逆转倍数分别为8.97,6.36,5.19和5.82.     

5-Aminolaevulinic acid-mediated photodynamic therapy in multidrug resistant leukemia cells

To verify if photodynamic therapy (PDT) could overcome multidrug resistance (MDR) when it it applied to eradicate minimal residual disease in patients with leukemia, we investigated the fluorescence kinetics of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) and the effect of subsequent photodynamic therapy on MDR leukemia cells, which express P-glycoprotein (P-gp), as well as on their parent cells. Evaluation of PpIX accumulation by flow cytometry showed that PpIX accumulated at higher levels in mdr-1 gene-transduced MDR cells (NB4/MDR) and at lower levels in doxorubicin-induced MDR cells (NOMO-1/ADR) than in their parent cells. A P-gp inhibitor could not increase PpIX accumulation. Measurement of extracellular PpIX concentration by fluorescence spectrometry showed that P-gp did not mediate the fluorescence kinetics of ALA-induced PpIX production. Assessment of ferrochelatase activity using high-performance liquid chromatography indicated that PpIX accumulation in drug-induced MDR cells was probably regulated by this enzyme. Assessment of phototoxicity of PDT using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that PDT was effective in NB4, NB4/MDR, NOMO-1 and NOMO-1/ADR cells, which accumulated high levels of PpIX, but not effective in K562 and K562/ADR cell lines, which accumulated relatively low levels of PpIX. These findings demonstrate that P-gp does not mediate the ALA-fluorescence kinetics, and multidrug resistant leukemia cells do not have cross-resistance to ALA-PDT.     

Analysis of oxidized multi-walled carbon nanotubes in single K562 cells by capillary electrophoresis with laser-induced fluorescence

Short oxidized multi-walled carbon nanotubes (CNT) were derivatized with fluorescein isothiocyanate (FITC). Capillary electrophoresis coupled with laser-induced fluorescence (CE–LIF) was then used to separate and detect the fluorescently labeled carbon-nanotube probes (CNTP) in multidrug-resistant cells (K562A) and the parent cells (K562S). Greater expression of P-glycoprotein in K562A cells than in K562S cells was confirmed by use of anti-P-glycoprotein antibody and flow-cytometric analysis. Analyses of CNTP in both cell lines using both CE–LIF and flow cytometry showed that CNTP could traverse the cellular membrane without being pumped out by P-glycoprotein. The CNTP distributed in both cell lines was analyzed at the single cell level and the results were compared with those from analysis of ten cells and of the lysate from bulk cells. The results revealed the CE–LIF method could be used for quantitative analysis of CNT in single cells in studies of drug delivery and multidrug resistance.      

Cytotoxic polyprenylated xanthones from the resin of Garcinia hanburyi

Thirteen xanthones (1-13) were isolated from the resin of Garcinia hanburyi. Among them, two new compounds (namely gaudichaudic acid, and isogambogenic acid, 1, 2), and one new natural product (deoxygaudichaudione A, 3) were identified on the basis of extensive spectral evidence including detailed 2D NMR data. Ten of these xanthones were tested for their cytotoxicities against human leukemia K562 (K562/S) and doxorubicin-resistant K562 (K562/R) cell lines, and showed similar inhibitory effects on both cell lines, suggesting that this group of polyprenylated xanthones might not be multidrug resistance (MDR) substrates.     

Multiplex analysis of tumor multidrug-resistance genes expression with photonic suspension array

An easy-operated suspension array based on silica colloidal crystal beads is developed for multiplex analysis of tumor multidrug-resistance genes expression, such as multidrug resistance 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1), and potentially single nucleotide polymorphism. In order to obtain high fluorescence intensity, controlled PCR was used to amplify targets at the samples pretreatment stage. By optimizing the conditions a hybridization procedure, which is similar to nucleic acids analysis with binary probes, was established. Small amounts of analytes 10(-19) M could be detected by the method. The K562 cell, human myeloma cell, and its multidrug-resistance string, adriamycin-selected P-glycoprotein-overexpressed K562/A02, were analyzed by using an established procedure to validate feasibility. Clinical blood samples were detected by our method and real-time PCR simultaneously to validate accuracy. Moreover, when combined with multiplex controlled PCR, the method successfully meets the requirements of multiplex analysis. Hence, the method presented is a good method for multiplex analysis of tumor multidrug-resistance genes expression.     

Preferential cytotoxicity for multidrug-resistant K562 cells using the combination of a photosensitizer and a cyanine dye

The cyanine dye 1,1',3,3,3',3'-hexamethylindodicarbocyanine iodide (HIDC) protects K562 leukemia cells from photodynamic membrane damage caused by cis-di(4-sulfonatophenyl)diphenylporphine (TPPS2) and 420 nm light. This wavelength of light is chosen because it is absorbed by TPPS2, but not by HIDC. The photodynamic system studied may be useful as a model for antineoplastic therapy. A subline of K562 leukemia (K562/DOX), expressing the multidrug-resistance (MDR) phenotype, is found to accumulate smaller amounts of HIDC than the parent cell line and thus has less photoprotection. In the absence of added HIDC, the K562/DOX cell line is more resistant to photodynamic cytotoxicity than the K562 cell line. The resistance of the K562/DOX cell line is not due to a smaller accumulation of TPPS2 than the K562 cell line. However, when both cell lines are incubated with HIDC and TPPS2, and then exposed to light, the K562/DOX cell line becomes more sensitive to photodynamic cell damage than the K562 cell line. The combination of a photosensitizer with a cationic or lysomorphotropic photoprotector represents a novel strategy for the eradication of malignant cells expressing the MDR phenotype.     

MEKC‐LIF analysis of rhodamine123 delivered by carbon nanotubes in K562 cells

Oxidized single‐walled carbon nanotubes (o‐SWNTs) were employed as the drug carriers to deliver the small molecules of Rhodamine123 (Rho123) into the K562 cells via physical adsorption. However, due to the fluorescence quenching of Rho123 on carbon nanotubes, the quantitative determination of Rho123 in cells is difficult. Based on the MEKC coupled with LIF detection, a quantitative approach was developed for the determination of Rho123 delivered into K562 cells by o‐SWNTs. Where the adsorbed Rho123 on o‐SWNTs could be desorbed by SDS in running buffer and be simultaneously separated with o‐SWNTs due to the differences of their electrophoretic mobility by applying the electric potential at the both ends of capillary. Using this approach, the intracellular uptakes of Rho123 in multidrug‐resistant and multidrug‐sensitive leukemia cells were quantified, and the results showed that o‐SWNTs could be used as the potential drug carriers to deliver small molecules into cells via the physical adsorption along with the circumventing of multidrug resistance of leukemia cells.     

The cellular labeling and pH-sensitive responsive-drug release of celastrol in cancer cells based on Cys-CdTe QDs

As one of the active compounds derived from Traditional Chinese Medicine,Celastrol(CSL)had cytotoxicity for human leukemia cancer cells K562 and its multidrug-resistant cell line K562/A02.Here,we introduced cysteamine-modified CdTe QDs as the labeling and drug carrier into CSL research and found that the self-assembly and conjugation of anticancer molecular CSL with the Cys-CdTe QDs could significantly increase the drug’s cytotoxicity for K562 cells.More important,these CSL-Cys-CdTe nanocomposites could overcome the multidrug resistance of K562/A02 cells and efficiently inhibit the cancer cell proliferation by realizing the pH-sensitive responsive release of CSL to cancer cells.The enhanced cytotoxicity was caused by the increase of the G2/M phase arrest for K562/A02 cells as well as for K562 cells.Cys-CdTe QDs can readily bind on the cell plasma membranes and be internalized into cancer cells to trace and detect human leukemia cancer cells in real time.In addition,these Cys-CdTe QDs can facilitate the inhibition of the multidrug resistance of K562/A02 cells and readily induce apoptosis.As a good photosensitizer for the therapy,labeling,and tracing of cancer cells,the combination of CSL with Cys-CdTe QDs can optimize the use of and a new potential therapy method for CSL and yield new tools to explore the mechanisms of active compounds from Traditional Chinese Medicine.     

Dielectrophoretic trapping of single leukemic cells using the conventional and compact optical measurement systems

Here, we report a microfluidic same‐single‐cell analysis to study the inhibition of multidrug resistance due to drug efflux on single leukemic cells. Drug efflux inhibition was investigated in the microfluidic chip using two different fluorescence detection systems, namely, a compact single‐cell bioanalyzer and the conventional optical detection system constructed from an inverted microscope and a microphotometer. More importantly, a compact signal generator was used to conduct dielectrophoretic cell trapping together with the compact SCB. By using the DEP force, a single acute myeloid leukemia cell was trapped in the cell retention structure of the chip. This allowed us to detect dye accumulation in the MDR leukemic cells in the presence of cyclosporine A (CsA). CsA and rhodamine 123 were used as the P‐glycoprotein inhibitor and fluorescent dye, respectively. The result showed that the Rh123 fluorescence signal in a single‐cell increased dramatically over its same‐cell control on both fluorescence detection systems due to the inhibition by CsA.     

Search for novel proteins involved in the development of chemoresistance in colorectal cancer and fibrosarcoma cells in vitro using two-dimensional electrophoresis, mass spectrometry and microsequencing.

In search of novel mechanisms that may lead to the development of chemoresistance of malignant tumors of the large bowel we used two-dimensional electrophoresis to identify proteins that were overexpressed in colorectal and fibrosarcoma cell lines that were resistant towards mitoxantrone. This cytostatic drug is known to lead to atypical multidrug resistance, i.e., the classical mechanism of multidrug resistance (MDR) accompanied by the overexpression of P-glycoprotein (P-gp) is ineffective. Using mass spectrometry and microsequencing we found adenine phosphoribosyl transferase and breast cancer specific gene 1 (BCSG1) overexpressed in the resistant colorectal tumor cell line. In the chemoresistant fibrosarcoma cell line we found two proteins that were overexpressed. One was identified as Rho-guanine dinucleotide phosphate (Rho-GDP) dissociation inhibitor and the other had sequence homologies with yeast protein yer-7. The putative role of these proteins is discussed.     

Ethyl lucidenates A reverses P-glycoprotein mediated vincristine resistance in K562/A02 cells

Multidrug resistance is a major unresolved obstacle to successful cancer chemotherapy. It is often associated with an elevated efflux of a variety of anticancer drugs by ATP-binding cassette transporters including P-glycoprotein, BCRP and MRP1. In this study, the reversal effect of Ethyl lucidenates A on K562/A02 cells was investigated. At concentrations of 10 μM, Ethyl lucidenates A could reverse the resistance of K562/A02 to vincristine up to 7.59 folds. Mechanistically, Ethyl lucidenates A could increase the intracellular accumulation of vincristine in K562/A02 cells through inhibiting the P-glycoprotein mediated drug-transport activity by rhodamine accumulation assay and cell cycle analysis. Further mechanistic investigation found that Ethyl lucidenates A did not alter P-glycoprotein expression. In conclusion, Ethyl lucidenates A could reverse the multidrug resistance of K562/A02 cells via its influence on P-glycoprotein drug-transport activity and thus, be a potential multidrug resistance reversal agent.     

An in vitro and in vivo study of a novel zinc complex, zinc N-(2-hydroxyacetophenone)glycinate to overcome multidrug resistance in cancer

Multiple drug resistance (MDR) remains a major clinical challenge for cancer treatment. P-glycoprotein is the major contributor and they exceed their role in the chemotherapy resistance of most of the malignancies. Attempts in several preclinical and clinical studies to reverse the MDR phenomenon by using MDR modulators have not yet generated promising results. In the present study, a co-ordination complex of zinc viz., Zn N-(2-hydroxyacetophenone)glycinate (ZnNG) has been synthesized, characterized and its antitumour activity was tested in vitro against drug sensitive and resistant human T-lymphoblastic leukemic cell lines (CCRF/CEM and CEM/ADR5000 respectively) and in vivo against Ehrlich ascites carcinoma (EAC) implanted in female Swiss albino mice. To evaluate the cytotoxic potential of ZnNG, we used sensitive CCRF/CEM and drug resistant CEM/ADR 5000 cell lines in vitro. Moreover, ZnNG also has the potential ability to reverse the multidrug resistance phenotype in drug resistant CEM/ADR 5000 cell line and induces apoptosis in combination with vinblastine. ZnNG remarkably increases the life span of Swiss albino mice bearing sensitive and doxorubicin resistant subline of EAC in presence and in absence of doxorubicin. In addition, intraperitoneal application of ZnNG in mice does not show any systemic toxicity in preliminary trials in normal mice. To conclude, a novel metal chelate of zinc viz., ZnNG, may be a promising therapeutic agent against sensitive as well as drug resistant cancers.     

Rapid determination of multidrug resistance-associated protein in cancer cells by capillary electrophoresis immunoassay

The adenosine triphosphate (ATP) binding-cassette (ABC) transporters are a superfamily of cellular proteins that have been partly implicated as a cause of multidrug resistance (MDR) in cancer cells. The ABC superfamily consists of P-glycoprotein, multidrug resistance-associated proteins (MRP) and breast cancer-related proteins, of which MRP is of particular interest because of its ability to efflux a broader range of substrates. Since MRP1 is the most prominent member of the MRP family, a simple technique is needed for its quantification. We developed a simple, fast (total analysis time of 3h) capillary electrophoresis immunoassay (CEIA) for the quantification of MRP1 in cancer cells. MRP1 antibody was labeled with fluorescein isothiocyanate. The labeled antibody was incubated with the cell lysate for a fixed interval (1h), after which the cell lysate mixture was directly injected into the capillary to separate the complex of MRP1 and its antibody from free antibody. The noncompetitive CEIA method had a limit of detection of 0.2 nM and a good linear range (1.7-14.9×10(4) cells), and was fairly reproducible (RSD<10%). The results showed that two cell lines, A549 and RDES, expressed MRP1 in the absence of doxorubicin (DOX), with A549 registering a higher expression. Compared to DOX-free cancer cells, there was an acceleration of MRP1 expression during the 12h-exposure to DOX, after which the level of expression remained nearly constant as the intracellular accumulation of DOX decreased. The results obtained in this work indicate that the developed CEIA method is useful for relative quantification of MRPs in cancer cells.     

N,N-二(8-黄酮甲基)香叶胺类化合物的合成及生理活性

设计合成了4个N,N-二（8-黄酮甲基）香叶胺类化合物,所有目标化合物的结构均经1H NMR、MS和元素分析测试技术确证。采用MTT法考察了目标化合物对K562（白血病细胞）和SMMC7721（肝癌细胞）2种肿瘤细胞的体外抑制活性,结果表明,所测化合物对2种肿瘤细胞都有体外抑制活性,其中N,N-二(3′,4′-二甲氧基-8-黄酮甲基）香叶胺(1c)的活性最好,IC50值分别为5.78和3.85 μmol/L,N,N-二（4′-氟-8-黄酮甲基）香叶胺(1a)和化合物1c对K562（白血病细胞）的体外抑制活性明显优于商品药物美法仑(Melphalan)。以溴化乙锭(EB)为荧光探针测定了化合物1c与鲱魚精DNA有较强的相互作用。     

Chemical constituents from Cicuta virosa Linnaeus and their reversal effects on doxorubicin-resistant human myelogenous leukemia (K562/A02) cells

Two new compounds,11,11 '-dimer of scopoletin(1) and 11-O-β-glucopyranosylhamaudol(2),together with seven known compounds were isolated and identified from the whole grass of Cicuta virosa.The chemical structures of the isolated compounds were elucidated using different spectroscopic methods.In addition,the chemical constituents were evaluated for multidrug resistance reversing activity towards doxorubicin-resistant K562/A02 cells.Compounds 1,8,and 9 were endowed with remarkable MDR reversing effects.     

Analysis of doxorubicin uptake in single human leukemia K562 cells using capillary electrophoresis coupled with laser-induced fluorescence detection

The doxorubicin (DOX) uptake in single human leukemia K562 cells with changes in both drug dosage and exposure period was studied using capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF) detection. The cells were treated with DOX at different concentrations (1, 3, 10, 20, 30, and 50 μM) and for different exposure times (1, 3, 5, 24, and 48 h). At least 20 cells were analyzed for each DOX-treated cell population. A marked heterogeneity in DOX uptake among single cells was observed, because the relative standard deviation of the uptake of DOX by single cells ranged from 24.0% to 61.1% within each cell population. The cell-to-cell heterogeneity in DOX uptake first decreased and then became constant with increasing drug concentration, but it did not exhibit regular variation with increasing exposure time. The mean DOX uptake was a linear function of drug concentration (r ≥ 0.9667). In terms of the correlation with exposure time, the mean DOX uptake reached its maximum at 3 h for the cell populations treated with 1–10 μM DOX, while it kept increasing during 48 h exposure of cell populations to 20–50 μM DOX. Because it eliminates DOX fluorescence quenching and sample loss, the CE-LIF method directly detects the true DOX uptake by single cells, and thus presents accurate information on both the cell-to-cell heterogeneity in DOX uptake and the patterns of DOX uptake in K562 cells as functions of drug concentration and exposure time.     

Design, synthesis and biological activity of 7-O-(4-O-acetyl-3-iodo-2,3,6-trideoxy-alpha-L-arabino-hexopyranosyl) daunomycinone and 7-O-(3-chloro-2,3,6-trideoxy-4-O-propanoyl-alpha-L-lyxo- hexopyranosyl)daunomycinone

The synthesis and pharmacological evaluation of 7-O-(4-O-acetyl-3-iodo-2,3,6-trideoxy-alpha-L-arabino-hexopyranosyl) daunomycinone and 7-O-(3-chloro-2,3,6-trideoxy-4-O-propanoyl-alpha-L-lyxo-hexopyrano syl) daunomycinone are described. Their cytotoxic activity was evaluated against normal and resistant cell lines. Both compounds exhibited activity against the adriamycin resistant cell line KB-A1. These results support the hypothesis that the increased lipophilicity of the sugar part of anthracyclines is associated with their ability to overcome multidrug resistance (MDR).     

Takaneones A-C, prenylated butylphloroglucinol derivatives from Hypericum sikokumontanum

Three new prenylated butylphloroglucinol derivatives, takaneones A-C (1-3), were isolated from the MeOH extracts of the aerial parts of Hypericum sikokumontanum together with two new prenylated phloroglucinol derivatives, takaneols A and B (4 and 5). The structures of the isolated compounds were elucidated by exhaustive spectroscopic analysis. The cytotoxicities of the isolated compounds against human cancer cell lines were evaluated. Compounds 2-4 showed cytotoxicities against K562/Adr multi-drug resistant (MDR) cancer cells with IC₅₀ values ranging from 4.7 to 10.0 μg/mL, which were slightly more potent than doxorubicin. Their potency of cytotoxicities against MDR cancer cell lines (KB-C2 and K562/Adr) were similar to those against sensitive cell lines (KB and K562).     

抗肿瘤Lamellarin类生物碱的合成研究新进展

Lamellarin类化合物是从海洋软体动物中分离得到的一类生物碱. Lamellarin类生物碱及其类似物具有良好的抑制肿瘤细胞增殖、逆转p-糖蛋白介导的多药耐药等活性, 显示出成为抗肿瘤候选药物的潜力. 近年来, 对Lamellarin类生物碱结构改造和新型杂合体及类似物的研究, 已成为新型抗肿瘤候选药物研究的热点之一. 综述了Lamellarin类生物碱的全合成策略与技术等方面的研究新进展.