Streamlined in vivo selection and screening of human prostate carcinoma avid phage particles for development of peptide based in vivo tumor imaging agents

Bacteriophage (phage) display has been exploited for the purpose of discovering new cancer specific targeting peptides. However, this approach has resulted in only a small number of tumor targeting peptides useful as in vivo imaging agents. We hypothesize that in vivo screening for tumor uptake of fluorescently tagged phage particles displaying multiple copies of an in vivo selected tumor targeting peptide will expedite the development of peptide based imaging agents. In this study, both in vivo selection and in vivo screening of phage displaying foreign peptides were utilized to best predict peptides with the pharmacokinetic properties necessary for translation into efficacious in vivo imaging agents. An in vivo selection of phage display libraries was performed in SCID mice bearing human PC-3 prostate carcinoma tumors. Eight randomly selected phage clones and four control phage clones were fluorescently labeled with AlexaFluor 680 for subsequent in vivo screening and analyses. The corresponding peptides of six of these phage clones were tested as 111In-labeled peptide conjugates for single photon emission computed tomography (SPECT) imaging of PC-3 prostate carcinomas. Two peptide sequences, G1 and H5, were successful as in vivo imaging agents. The affinities of G1 and H5 peptides for cultured PC-3 cells were then analyzed via cell flow cytometry resulting in Kd values of 1.8 μM and 2.2 μM, respectively. The peptides bound preferentially to prostate tumor cell lines compared to that of other carcinoma and normal cell lines, and H5 appeared to possess cytotoxic properties. This study demonstrates the value of in vivo screening of fluorescently labeled phage for the prediction of the efficacy of the corresponding 111In-labeled synthetic peptide as an in vivo SPECT tumor imaging agent.     

Enhanced fluorescence images for labeled cells on silver island films

Silver island films (SIFs) were deposited on glass substrates to serve as supports. T-Lymphocytic (PM1) cell lines were labeled by Alexa Fluor 680-dextran conjugates on the membranes or by YOYO in the nuclei. The fluorescence images of the cell lines were recorded in the emission intensity and lifetime using scanning confocal microscopy. The fluorescence signals by the fluorophores bound on the cell membranes were enhanced significantly by SIF supports as compared with those on the glass. In addition to the increase in the intensity, there was a dramatic shortening of the emission lifetime. In contrast to the Alexa Fluor 680 fluorophores on the membranes, the YOYO fluorophores intercalated in the cell nuclei were not influenced significantly by the silver islands. This result can be interpreted by an effect of the distance on coupling between the fluorophores and metal particles: the fluorophores on the cell membranes are localized within, but the fluorophores in the cell nuclei are beyond the region of metal-enhanced fluorescence. Thus, the metal supports can be used to improve the detection sensitivity for target molecules on cell surfaces when they are fluorescently labeled.     

Syntheses and Antiproliferative Activities of Novel Diarylthiosemicarbazide Derivatives

A series of novel N-methylpicolinamide-moiety containing diarylthiosemicarbazide derivatives was prepared and evaluated for their in vitro antiproliferative activity against three cancer cell lines(human alveolar epithelial cell A549, human lung cancer cell H460 and human colorectal cancer cell HT-29) by 3-(4,5-dimethyl)thiazolyl-diphenyltetrazoliumromide(MTT) assay. Six compounds(7b―7g) with halogen substituents exhibited preferable cytotoxicity against one or more cell lines in a low micromolar range. Especially, the most promising compound 7g exhibited remarkable antiproliferative activity with the IC50 values of 2.2, 1.8 and 5.2 μmol/L against A549, H460 and HT-29 cell lines respectively, which is comparable to sorafenib.     

嘌呤磺胺衍生物的合成及抗肿瘤活性

合成了一系列结构全新的嘌呤磺胺类衍生物, 并应用溴化噻唑蓝四氮唑(MTT)法进行了初步体外抗肿瘤细胞增殖活性研究. 结果表明, 嘌呤环C2位、 N6位和N9位的取代对活性均有较大影响, C2位引入苯磺酰基哌嗪片段后有利于提高抗肿瘤活性. 化合物17d对3株肿瘤细胞PC-3, HCT116和K562 的增殖均有明显抑制作用, 且强度与阳性对照药Roscovitine相近.     

Design,Synthesis and Antiproliferative Activities of Diaryl Thiourea Derivatives as Anticancer Agents

Two new series of diaryl thiourea containing sorafenib derivatives 9a – 9t were designed and synthesized, and their antiproliferative activities against PC‐3, HCT116 and MDA‐MB‐231 cell lines were evaluated. All compounds generally showed antiproliferative activity to PC‐3 cells, most of the analogs exhibited potent antiproliferative activity to HCT116 cells, and compounds 9e , 9f , 9o and 9p demonstrated inhibitory activities against all three cell lines. The structures of all the newly synthesized compounds were determined by ¹H NMR, ¹³C NMR and HRMS.     

Encapsulation of fluorescent molecules by functionalized polymeric nanocontainers: investigation by confocal fluorescence imaging and fluorescence correlation spectroscopy

Nanocontainers (NCs) were prepared from amphiphilic triblock copolymers, having an average molecular weight of around 8000 g/mol, by using previously published preparation methods consisting of dispersing the polymer in an aqueous buffer solution containing molecules for encapsulation. A small molecular weight fluorophore, sulforhodamine B, as well as the fluorescent protein avidin labeled with Alexa 488 were encapsulated, and the resulting nanocontainers were characterized using fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS). Nanocontainer size determination by FCS is very robust and compares well with results obtained from photon correlation spectroscopy: the measured diameters of the polymeric nanocontainers vary between 140 and 172 nm. Encapsulation of fluorescent molecules was determined by evaluating the molecular brightness of nanocontainers with an encapsulated fluorescently labeled protein (avidin-Alexa 488). Results indicate that the number of encapsulated avidin-Alexa 488 molecules corresponds well with the initial concentration of the fluorescently labeled protein and the encapsulated volume. A nanocontainer binding assay was developed using biotinylated fluorescently labeled nanocontainers. Binding of biotinylated nanocontainers to fluorescently labeled streptavidin was followed by fluorescence cross-correlation spectroscopy. The intrinsic dissociation constant, K(d), of labeled streptavidin to the ligand-modified nanocontainers is 1.7 +/- 0.4 x 10(-8) M, and about 1921 +/- 357 molecules of labeled streptavidin are bound to each nanocontainer.     

Synthesis and antiproliferative activities of thioxoflavonoids on three human cancer cells

Two series of fifteen novel thioxoflavonoids 2a-2h and 4a-4g were synthesized from corresponding flavonoids 1a-1h and 3a-3g by reacting with Lawesson’s reagent, respectively. Their in vitro antiproliferative activities were evaluated on a panel of three human cancer cell lines (Hela, HCC1954 and SK-OV-3) using cell counting kit-8 (CCK-8) assay. The results showed that most of the target compounds exhibited moderate to good antiproliferative activities against the three human cancer cell lines. In particular, thioxoflavonoids 2f and 2g showed the strongest antiproliferative activity on all three human cancer cell lines with IC₅₀ values ranging from 3.34 to 4.67 μM, 4f showed the best antiproliferative activity on Hela cells (IC₅₀ 2.30 μM), 2e showed the best antiproliferative activity on HCC1954 cells (IC₅₀ 2.13 μM) and SK-OV-3 cells (IC₅₀ 2.33 μM). The antiproliferative activities may be involved in their antioxidant activity, which can be speculated by their ability to scavenge free radicals and by their capacity of affecting key redox enzymes.     

Hydroxylated oxanes as xyloside analogs for determination of the minimal binding requirements of β4GalT7

β-1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme in the biosynthesis of glycosaminoglycan (GAG) chains. Natural and synthetic xylosides can be used to both inhibit and prime GAG synthesis by acting as inhibitors or substrates for β4GalT7. In this report, we exploit hydroxylated oxanes as deoxygenated xyloside analogs to clarify the minimum protein-ligand interactions required for galactosylation and/or inhibition. Enantiomerically pure substances were synthesized using a chiral pool approach whereas the corresponding racemates were obtained from simple starting materials. The results of a β4GalT7 assay show that a single hydroxyl group on an oxane ring is insufficient to induce galactosylation or inhibition, which implies that at least two substituents, one of which being 3-OH, needs to be present.     

Synthesis,characterization, and biological evaluations of some steryl 2-methoxybenzoates as anticancer agents

Using cholesterol, stigmasterol and sitosterol as starting materials, a series of 7-subsitituted-ster-3-yl 2-methoxybenzoate analogs were prepared through reacting with 2-methoxybenzoyl chloride and introducing some function groups, such as carbonyl, hydroxyl and various thiosemicarbazones, at 7-position of steroidal nucleus. The structures of these new compounds were characterized by IR, NMR and HRMS. Their antiproliferative activities were evaluated by using several types of cancer cells. Interestingly, the compounds displayed potent antiproliferative activity against CNE-2 (nasopharyngeal carcinoma cell lines), BEL-7402 (human liver cancer cell lines) and HepG2 (human liver cancer cell lines), suggesting that they have potential to be drug candidates for cancer treatment.     

Physangulidines A, B, and C: three new antiproliferative withanolides from Physalis angulata L

Bioassay-directed fractionation of the whole plant of Physalis angulata L. afforded three new antiproliferative withanolides with an unusual carbon framework: physangulidines A (1), B (2), and C (3). Structures of the three isomeric withanolides were determined by a combination of HRMS, NMR spectroscopic, and X-ray crystallographic methods. Each has shown significant antiproliferative activity against DU145 prostate cancer cells. Physangulidine A (1) was further tested against a wide range of additional cancer cell lines and found to exhibit significant antiproliferative activity.     

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.      

Enhancement of gemcitabine against pancreatic cancer by loading in mesoporous silica vesicles

Gemcitabine(Gem) is currently the first-line chemotherapeutic drug in management of pancreatic cancer, however the therapeutic efficacy of Gem is limited due to its short half-life and poor cell membrane permeability. Here we designed mesoporous silica vesicles(MSVs) with large pore sizes as a novel drug delivery system. The MSVs were synthesized using cetyltrimethyl ammonium bromide(CTAB) as a structure-directing agent, tetraethoxysilane(TEOS) as silica source in n-hexane/water biliquid system. By virtue of the large pore size and large pore volume of the MSVs, Gem was loaded into the mesoporous of MSVs via "nanocasting" method. In vitro drug release experiments of gemcitabineloaded MSVs showed an accelerating release of gemcitabine in acidic condition. These fluorescently labeled MSVs could be effectively internalized by both a human(BxPC-3) and a mouse pancreatic cancer cell lines(Pan02). Additionally, some MSVs could even reach the nuclei of the pancreatic cancer cells. Cell viability assays demonstrated that gemcitabine-loaded MSVs exhibited enhanced anticancer activity in inhibiting the proliferation of Bx PC-3 and Pan02 cells compared with free Gem, while the MSVs alone showed no significant cytotoxicity. Our results indicate that our synthesized MSVs might represent a promising novel drug delivery platform for the treatment of pancreatic cancer.     

Design and Synthesis of Hydrazine and Oxadiazole-containing Derivatives of Sorafenib as Antitumor Agents

A series of hydrazine and oxadiazole analogs of Sorafenib was designed, synthesized and characterized by proton nuclear magnetic resonance(¹H NMR) spectrometry and high resolution mass spectrometry(HRMS). The antiproliferative activities of these compounds against human colorectal carcinoma(HCT-116) and human breast cancer (MDA-MB-231) tumor cell lines were evaluated in vitro by MTT method[MTT=3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide]. The bioassay results suggest that most of the synthesized compounds have antitumor potential to HCT-116 cell line compared with MDA-MB-231 cell line. Compounds 8a,8b,8d, 8e,9f and 9j competitive with Sorafenib demonstrated antiproliferative activities on HCT-116 cell line.     

查尔酮曼尼希碱类衍生物的设计、合成及抗肿瘤活性研究

以丹皮酚和对氯苯甲醛为起始原料,通过Claisen-Schmidt反应得到2-羟基-4-甲氧基-4′-氯查尔酮(3),再经过Mannich反应得到10个查尔酮曼尼希碱衍生物(4a～4e, 5a～5e)。目标化合物结构均经高分辨质谱、核磁共振氢谱、碳谱进行确证。采用MTT法测试了所合成化合物对人肺癌细胞A549、人肝癌细胞HepG2的体外抗增殖活性。结果表明,目标化合物对肿瘤细胞A549、HepG2均具有较强的细胞增值抑制作用,且明显优于阳性对照药5-氟尿嘧啶。     

N9位芳基取代嘌呤-8-酮类衍生物的合成及抗肿瘤活性

合成了一系列N9位芳基取代嘌呤-8-酮类衍生物, 利用核磁共振氢谱(¹H NMR)、 核磁共振碳谱(¹³C NMR)和高分辨质谱(HRMS)进行了结构确证. 采用四甲基偶氮唑盐(MTT)法测定了目标化合物的体外抗肿瘤细胞增殖活性. 结果表明, 嘌呤酮环的C2位及N9位的取代对活性有较大影响, C2位引入对位由含氮六元环取代的苯胺, N9位引入对三氟甲基苯均有利于提高抗肿瘤活性. 化合物12c对人白血病细胞(K562)、 人前列腺癌细胞(PC-3)、 人乳腺癌细胞(MDA-MB-231)及人结肠癌细胞(HCT116)的抑制效果明显优于阳性对照药R-Roscovitine.     

Distribution of glycoconjugates in mouse fibroblasts with varying degrees of tumorigenicity

Analysis of glucosamine labeled glycoconjugates in cultured cells has been made comparing 2 clones and the parent embryonic mouse cell line. Hyaluronic acid, heparan sulphate, and chondrotin sulphate as well as a complex mixture of glycopeptides were found in the medium, the trypsinate, and the trypsinized cells, although the distribution was not uniform. The 3 cell lines had very similar in vitro growth properties, including their plating efficiency in viscous medium. However, the tumorigenicity of the cells, determined syngeneic mice, was found to differ. All 3 cell lines were found to have similar glycoconjugate distributions, although a slight relative increase in labeled hyaluronic acid was found in the more tumorigenic mass cell line than either of the clones. The possible significance of this increase is discussed.     

Novel Nitric Oxide Donor Dinitroazetidine-Coumarin Hybrids as Potent Anti-Intrahepatic Cholangiocarcinoma Agents

Intrahepatic cholangiocarcinoma (iCC) is a serious liver cancer threatening human health. However, there are a few chemotherapeutic drugs for the treatment of iCC in the clinic. It is extremely urgent to develop new drugs for iCC. In this study, twenty dinitroazetidine and coumarin hybrids were synthesized and evaluated anti-iCC bioactivity as a new type of nitric oxide (NO) donors. Among them, compounds 2–5 and 21 showed a higher antiproliferative activity against RBE cell lines (human intrahepatic cholangiocarcinoma cell lines) and low cytotoxicity in nontumor cells (HOSEpiC and T29). The preliminary study of pharmacology mechanism indicated that compounds 2–5 and 21 could release effective concentration of NO in RBE cell lines, which leaded to inhibit the proliferation of RBE cell lines. The research results revealed that compound 3 inhibited the proliferation of RBE cell lines by inducing apoptosis and arresting cell cycle at G₂/M phase. Additionally, compound 3 had acceptable metabolic stability. Therefore, compound 3 was merited to further explore for developing a desirable NO donor lead with anti-iCC activity.     

Antiproliferative, antimicrobial and apoptosis inducing effects of compounds isolated from Inula viscosa

The antiproliferative and antimicrobial effects of thirteen compounds isolated from Inula viscosa (L.) were tested in this study. The antiproliferative activity was tested against three cell lines using the MTT assay. The microdilution method was used to study the antimicrobial activity against two Gram positive bacteria, two Gram negative bacteria and one fungus. The apoptotic activity was determined using a TUNEL colorimetric assay. Scanning electron microscopy was used to study the morphological changes in treated cancer cells and bacteria. Antiproliferative activity was observed in four flavonoids (nepetin, 3,3'-di-O-methylquercetin, hispidulin, and 3-O-methylquercetin). 3,3'-di-O-Methylquercetin and 3-O-methylquercetin showed selective antiproliferative activity against MCF-7 cells, with IC(50) values of 10.11 and 11.23 μg/mL, respectively. Both compounds exert their antiproliferative effect by inducing apoptosis as indicted by the presence of DNA fragmentation, nuclear condensation, and formation of apoptotic bodies in treated cancer cells. The antimicrobial effect of Inula viscosa were also noticed in 3,3'-di-O-methylquercetin and 3-O-methyquercetin that inhibited Bacillus cereus at MIC of 62.5 and 125 μg/mL, respectively. Salmonella typhimurium was inhibited by both compounds at MIC of 125 μg/mL. 3,3'-di-O-Methylquercetin induced damage in bacterial cell walls and cytoplasmic membranes. Methylated quercetins isolated from Inula viscosa have improved anticancer and antimicrobial properties compared with other flavonoids and are promising as potential anticancer and antimicrobial agents.     

Effects of Vanadyl Complexes with Acetylacetonate Derivatives on Non-Tumor and Tumor Cell Lines

Vanadium has a good therapeutic potential, as several biological effects, but few side effects, have been demonstrated. Evidence suggests that vanadium compounds could represent a new class of non-platinum, metal antitumor agents. In the present study, we aimed to characterize the antiproliferative activities of fluorescent vanadyl complexes with acetylacetonate derivates bearing asymmetric substitutions on the β-dicarbonyl moiety on different cell lines. The effects of fluorescent vanadyl complexes on proliferation and cell cycle modulation in different cell lines were detected by ATP content using the CellTiter-Glo Luminescent Assay and flow cytometry, respectively. Western blotting was performed to assess the modulation of mitogen-activated protein kinases (MAPKs) and relevant proteins. Confocal microscopy revealed that complexes were mainly localized in the cytoplasm, with a diffuse distribution, as in podocyte or a more aggregate conformation, as in the other cell lines. The effects of complexes on cell cycle were studied by cytofluorimetry and Western blot analysis, suggesting that the inhibition of proliferation could be correlated with a block in the G2/M phase of cell cycle and an increase in cdc2 phosphorylation. Complexes modulated mitogen-activated protein kinases (MAPKs) activation in a cell-dependent manner, but MAPK modulation can only partly explain the antiproliferative activity of these complexes. All together our results demonstrate that antiproliferative effects mediated by these compounds are cell type-dependent and involve the cdc2 and MAPKs pathway.     

Synthesis and Biological Evaluation of Novel Allobetulon/Allobetulin–Nucleoside Conjugates as AntitumorAgents

Allobetulin is structurally similar tobetulinic acid, inducing the apoptosis of cancer cells with low toxicity. However, both of them exhibited weak antiproliferation against several tumor cell lines. Therefore, the new series of allobetulon/allobetulin–nucleoside conjugates 9a–10i were designed and synthesized for potency improvement. Compounds 9b, 9e, 10a, and 10d showed promising antiproliferative activity toward six tested cell lines, compared to zidovudine, cisplatin, and oxaliplatin based on their antitumor activity results. Among them, compound 10d exhibited much more potent antiproliferative activity against SMMC-7721, HepG2, MNK-45, SW620, and A549 human cancer cell lines than cisplatin and oxaliplatin. In the preliminary study for the mechanism of action, compound 10d induced cell apoptosis and autophagy in SMMC cells, resulting in antiproliferation and G0/G1 cell cycle arrest by regulating protein expression levels of Bax, Bcl-2, and LC3. Consequently, the nucleoside-conjugated allobetulin (10d) evidenced that nucleoside substitution was a viable strategy to improve allobetulin/allobetulon’s antitumor activity based on our present study.