Passage-restricted differentiation potential of mesenchymal stem cells into cardiomyocyte-like cells

Mesenchymal stem cells (MSCs) have limited ability to differentiate into cardiomyocytes and the factors affect this process are not fully understood. In this study, we investigated the passage (P)-related transdifferentiation potential of MSCs into cardiomyocyte-like cells and its relationship to the proliferation ability. After 5-azacytidine treatment, only P4 but not P1 and P8 rat bone marrow MSCs (rMSCs) showed formation of myotube and expressed cardiomyocyte-associated markers. The growth property analysis showed P4 rMSCs had a growth-arrest appearance, while P1 and P8 rMSCs displayed an exponential growth pattern. When the rapid proliferation of P1 and P8 rMSCs was inhibited by 5-bromo-2-deoxyuridine, a mitosis inhibitor, only P1, not P8 rMSCs, differentiated into cardiomyocyte-like cells after 5-azacytidine treatment. These results demonstrate that the differentiation ability of rMSCs into cardiomyocytes is in proliferation ability-dependent and passage-restricted patterns. These findings reveal a novel regulation on the transdifferentiation of MSCs and provide useful information for exploiting the clinical therapeutic potential of MSCs.     

氨基修饰的纳米纤维对人和大鼠 MSCs增殖分化的影响

目的:探讨氨基修饰后的静电纺丝纳米纤维对大鼠和人骨髓来源的间充质干细胞(Rat and human bone marrow mesenchymal stem cells, r MSCs and hMSCs)增殖及成骨分化的影响。方法:采用静电纺丝法制备聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)纳米纤维,用氨气等离子体处理其表面来接枝氨基;通过测量PLGA纳米纤维(NF)及氨基修饰后的纳米纤维(NF-NH_2)接触角来证明修饰效果;将r MSCs和hMSCs分别接种于NF和NF-NH_2,用CCK-8试剂盒检测接种后1, 3 (4), 7天的细胞增殖;接种后的21天,用茜素红S染色(ARS)法检测细胞成骨分化情况。结果:氨气等离子体处理后纳米纤维接触角从81.28±0.33降低至53.99±0.79,说明氨基修饰后的PLGA NF亲水性增加;CCK-8结果显示氨基修饰增加了r MSCs的黏附,接种24 h后r MSCs在NF和NF-NH_2上的检测吸光值分别为0.096±0.011和0.175±0.014(P0.001),而对hMSCs黏附和增殖没有影响,接种24 h后hMSCs在NF和NF-NH_2上的检测吸光值分别为0.237±0.004和0.238±0.006(P0.05);ARS染色结果显示氨基修饰后r MSCs成骨分化增多(在NF和NF-NH_2表面ARS染色区域比例分别13.147±3.223%和36.677±5.230%),而hMSCs在修饰前后的纳米纤维上均有表达(修饰前后ARS染色比例分别为50.283±2.942%和38.254±3.272%)。结论:氨基修饰的NF可以促进大鼠来源的MSCs黏附增殖以及成骨分化,而对人骨髓来源的MSCs没有显著影响,这提示我们MSCs的增殖分化行为可能具有种属依赖性。     

Synthesis, biological activity, and SAR of antimycobacterial 2- and 8-substituted 6-(2-furyl)-9-(p-methoxybenzyl)purines

A number of 6-(2-furyl)-9-(p-methoxybenzyl)purines carrying a variety of substituents in the 2- or 8-position have been synthesized and their ability to inhibit growth of Mycobacterium tuberculosis in vitro has been determined. It is demonstrated that sterical hindrance in the purine 8-position reduces activity and that C-8 should be unsubstituted. In the purine 2-position small, hydrophobic substituents are beneficial. The electronic properties of the 2-substituents appear to have only a minor influence on bioactivity. The compounds studied exhibit low toxicity toward mammalian cells (VERO cells) and are essentially inactive toward Staphylococcus aureus and Escherichia coli. The most active and selective antimycobacterial in the series detected to date is the novel 2-methyl-6-furyl-9-(p-methoxybenzyl)purine with MIC=0.20 microg/mL against M. tuberculosis and IC(50) against VERO cells >62.5 microg/mL. Also the novel 2-fluoro analog and the previously known 2-chloro compound, both with MIC=0.39 microg/mL, are highly interesting drug candidates.     

Therapeutic properties and molecular docking study of some phenolic compounds as anti-human lung cancer potential: A biochemical approach

Chloroxine (5,7-dichloro-8-hydroxyquinoline) is a molecule utilized in some shampoos for the therapy of seborrheic dermatitis of the scalp and dandruff. In this study, we investigated the inhibition effects of 5,7-dichloro-8-hydroxyquinoline and methyl 3,4,5-trihydroxybenzoate compounds on the 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA Reductase) and urease enzymes. We have obtained results for the HMG-CoA Reductase and urease enzymes at the micromolar level. In our study, inhibition result of 5,7-dichloro-8-hydroxyquinoline and Methyl 3,4,5-trihydroxybenzoate on HMG-CoA reductase showed lower values 2.28 ± 0.78 and 33.25 ± 5.04 µg/ml, respectively. Additionally, inhibition result of 5,7-dichloro-8-hydroxyquinoline and methyl 3,4,5-trihydroxybenzoate on urease showed lower values 6.18 ± 1.38 and 8.51 ± 1.35 µg/ml, respectively. Molecular docking calculations were made for their biological activities were compared. In the present work, the structures of the related compounds ( 1 and 2 ) were drawn using Gaussian 09 software and done geometry optimization at DFT/B3LYP/6-31G* basis set with aforementioned program. Cytotoxicity potential of these compounds against human lung cancer demonstrated that these compounds had good cytotoxic effects. Both compounds significantly decreased lung cell viability from low doses. In addition, 100 µM dose of all compounds caused significant reductions in lung cell viability. In general, we can say that of the two tested compounds, 5,7-dichloro-8-hydroxyquinoline and methyl 3,4,5-trihydroxybenzoate have cytotoxic effects in all cell types, and this effect is particularly strong in lung cells. Activities were performed at concentrations of 10, 20, 50, 70, and 100 µl and we achieved good results. Lung cell viability (%) value was better at 100 µl concentration and IC₅₀ of them were 54.28 and 48.05 µM.     

Biological evaluation of newly synthesized quinoline-5,8-quinones as Cdc25B inhibitors

Cdc25B protein phosphatase represents an attractive potential therapeutic target for small molecule intervention because of its central role in positively regulating cyclin dependent kinases and thus cell proliferation, as well as its elevated levels observed in many human tumors. Among the most potent previously identified Cdc25 inhibitors have been quinoline quinones, which have a rich legacy as therapeutic agents but have also been associated with nonspecific interactions. In this study, we have interrogated the structure-activity relationship of a focused series of C2-, C3-, or C4-modified quinoline-5,8-quinones on Cdc25B inhibition in vitro. Substitution at the C3-position in this small chemical series were slightly superior to substitutions at the C3-position. For all compounds, recombinant human Cdc25B was approximately 5-fold more sensitive compared to recombinant human PTP1B. Two compounds inhibited HeLa cell growth with IC50 values of approximately 2 microM. Consistent with other para-quinones, some members of this series generated intracellular reactive oxygen species and the in vitro enzyme inhibition was mitigated by addition of reductants or catalase. These results indicate that chemical modifications on the pyridine core are tolerated, providing additional sites for future structural modification of this biologically active pharmacophore.     

Differentiation of adult rat bone marrow stem cells into epithelial progenitor cells in culture

We have previously obtained monoclonal bone marrow stem cells from adult rats (rMSCs) and induced them into phenotypic neurons. In the present study, we aimed to induce rMSCs into epithelial cells by culturing them onto compartmentalized permeable supports, which have been used for growing a variety of polarized epithelia in culture. Hematoxylin staining showed that after 4 days grown on permeable supports, rMSCs formed an epithelial-like monolayer. Immunofluorescence of the permeably-supported monolayers, but not the rMSCs grown in culture flasks, showed positive signals for epithelial markers, cytokeratin 5 & 8. RT-PCR results also showed the mRNA expression of epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) as well as tight junction protein ZO-1 in the rMSC-derived monolayers grown on permeable supports but absent from those grown in culture flasks. However, western blot only detected protein expression of ZO-1 but not ENaC nor CFTR. The short-circuit current measurements showed that the rMSC-derived monolayers grown on permeable supports exhibited a trans-monolayer resistance of 30-50 Omega cm(2); however, the monolayers did not respond to activators or blockers of CFTR or ENaC. The results suggest that compartmentalized or polarized culture conditions provide a suitable environment for rMSCs to differentiate into epithelial progenitor cells with tight junction formation; however, this condition is not sufficient for functional expression of epithelial ion channels associated with well-differentiated epithelia.     

9-Benzylpurines with inhibitory activity against Mycobacterium tuberculosis

9-Benzylpurines with a variety of substituents in the 2-, 6- and/or 8-position have been prepared and screened for antimycobacterial effects. High inhibitory activity against Mycobacterium tuberculosis was found for 9-benzylpurines carrying a phenylethynyl-, trans-styryl or aryl substituents in the 6-position and generally chlorine in the 2-position tends to increase activity.     

Five-coordinated rhodium(I) carbonyl compounds

The title compounds were synthesised by the replacement of chlorine in Rh(CO)(PPh₃)₂Cl with monobasic bidentate chelating ligands such as salicylaldehyde, acetylacetone, benzoylacetone, dibenzoylmethane, 8-hydroxyquinoline, benzoylphenyl hydroxylamine, 2-hydroxyacetophenone and 2-hydroxybenzophenone. IR spectral evidence points out that these compounds have a trigonal bipyramidal geometry around rhodium in the solid state. However, in benzene solutions, except for the 8-hydroxyquinoline and 2-hydroxybenzophenone derivatives they all take a square planar structure, as seen from their electronic spectra.     

Synthesis and biological evaluation of 4'-(6,7-disubstituted-2,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ol as potent Chk1 inhibitors

A new series of potent tricyclic pyrazole-based Chk1 inhibitors are described. Analogues disubstituted on the 6- and 7-positions show improved Chk1 inhibition potency compared with analogues with a single substituent on either the 6- or 7-position. Based on the lead compound 4'-(6,7-dimethoxy-2,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ol (2), detailed SAR studies on the 6- and 7-positions were performed. 3'-morpholin-4'-yl-propoxy, pyridin-4'-ylmethoxy, pyridin-3'-ylmethoxy, 2'-(5'-ethyl-pyridin-2'-yl)-ethoxy, pyridin-2'-ylethoxy, (6'-methyl-pyridin-2'-yl)-propoxyethoxy, 2',3'-dihydroxyl-1'-yl-propoxy, and tetrahydro-furan-3'-yloxy have been identified as the best groups on the 6-position when the 7-position is substituted with methoxyl group. Pyridin-2'-ylmethoxy and pyridin-3'-ylmethoxy have been identified as the best substituents at the 7-position while the 6-position bearing methoxyl group. These compounds significantly potentiate the cytotoxicity of DNA-damaging antitumor agents in a cell-based assay and efficiently abrogate the doxorubicin-induced G2/M and the camptothecin-induced S checkpoints, suggesting that their potent biological activities are mechanism-based through Chk1 inhibition.     

Plasticity of rat bone marrow-derived 5-hydroxytryptamine-sensitive neurons: dedifferentiation and redifferentiation

Inducing cellular dedifferentiation has been proposed as a potential method for enhancing endogenous regeneration in mammals. Here we demonstrate that phenotypic and functional neurons derived from adult rat bone marrow stromal stem cells (MSCs) can be induced to undergo dedifferentiation, then proliferation and redifferentiation. In addition to morphological changes and expression of neuronal markers, neuron-specific enolase and neurofilament H, functional differentiation was monitored by intracellular Ca2+ mobilization in response to a ubiquitous neurotransmitter, 5-hydroxytryptamine (5-HT) at different stages. The neurons derived from rMSCs were found to have increased 5-HT response. This 5-HT sensitivity could be reversed to basal level similar to that found in rMSCs when neurons, up to 3 days after neuronal induction, were induced to undergo dedifferentiation. Increase in 5-HT-induced Ca2+ mobilization was again observed when rMSCs derived from dedifferentiated neurons were induced to redifferentiate into neurons again. Variation in 5-HT1A receptor immunoreactivity was observed in stem cells, differentiated neurons, dedifferentiated neurons and redifferentiation neurons, consistent with their respective 5-HT sensitivity. These results suggest that adult bone marrow-derived 5-HT sensitive neurons are capable of dedifferentiation, then proliferation and redifferentiation, indicating their plasticity and potential use in treatment of neural degenerative diseases.     

黄芩素的结构修饰以及抗肿瘤活性研究

为了改善黄芩素的抗肿瘤活性。本实验以黄芩素为原料,对其进行结构修饰。首先通过mannich反应,在8位引入胺亚甲基,然后通过酰基化反应在7位(6位)酚羟基上引入不同的疏水性基团。并利用CCK-8法对目标化合物进行抗MCF-7肿瘤细胞的活性评价。结果合成得到了6个目标化合物,通过1HNMR、13CNMR、MS和化学手段相结合的方法确定了其结构,其中化合物2~6为新化合物。实验利用黄酮类邻二酚羟基的特性,通过与氯化锶的络合反应,巧妙而简单的确证了所得目标化合物的酯键是在化合物的7位羟基上。抗MCF-7肿瘤活性实验表明,在黄芩素8位上引入含氮原子的胺亚甲基后活性比先导化合物黄芩素强,在其7位上再引入酯键后3个化合物活性比先导化合物强。     

Enhanced FTase activity achieved via piperazine interaction with catalytic zinc

Benzocycloheptapyridine tricyclic compounds with piperazine or substituted piperidine moieties extending either from the 5- or 6-position of the tricyclic bridgehead exhibited enhanced FTase activity: this resulted from favorable binding of the ligand nitrogen with the catalytic zinc found in the FTase. A single isomer at C-11 with piperazine adduct extending from the 6-position, compound 24, exhibited excellent FTase activity with IC50 = 0.007 microM, soft agar IC50 = 72 nM, and Rat AUC(PO, 10 mpk) = 4.0 microM x h. X-ray of (-)-[8-chloro-6-(1-piperazinyl)-1H-benzo[5,6]]cyclohepta[1,2-b]pyridine-11-yl]-1-(methylsulfonyl)piperidine 24 bound to Ftase revealed favorable interaction between piperazine nitrogen and catalytic zinc atom.     

Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 2. Structure-activity relationships of the 7- and 5-, 6-, 8-positions

As a continuation of our efforts to discover and develop the apoptosis inducing 4-aryl-4H-chromenes as novel anticancer agents, we explored the SAR of 4-aryl-4H-chromenes with modifications at the 7- and 5-, 6-, 8-positions. It was found that a small hydrophobic group, such as NMe2, NH2, NHEt, and OMe, is preferred at the 7-position. Di-substitution at either the 5,7-positions or the 6,7-positions generally led to a large decrease in potency. Di-substitution at the 7,8-positions, in general, was found to result in potent compounds. 7-NMe2, 7-NHEt, 7-OMe, and 7,8-di-NH2 analogs were found to have similar SAR for the 4-aryl group, and several 7-substituted and 7,8-di-substituted analogs were found to have similar potencies as the lead compound MX58151 (2a) both as caspase activators and inhibitors of cell proliferation.     

Structure-dependent inhibition of bladder and pancreatic cancer cell growth by 2-substituted glycyrrhetinic and ursolic acid derivatives

Derivatives of oleanolic acid, ursolic acid and glycyrrhetinic acid substituted with electron-withdrawing groups at the 2-position in the A-ring which also contains a 1-en-3-one structure are potent inhibitors of cancer cell growth. In this study, we have compared the effects of several 2-substituted analogs of triterpenoid acid methyl esters derived from ursolic and glycyrrhetinic acid on proliferation of KU7 and 253JB-V bladder and Panc-1 and Panc-28 pancreatic cancer cells. The results show that the 2-cyano and 2-trifluoromethyl derivatives were the most active compounds. The glycyrrhetinic acid derivatives with the rearranged C-ring containing the 9(11)-en-12-one structure were generally more active than the corresponding 12-en-11-one isomers. However, differences in growth inhibitory IC(50) values were highly variable and dependent on the 2-substituent (CN vs CF(3)) and cancer cell context.     

Synthesis and pharmacology of pyrido[2,3-d]pyrimidinediones bearing polar substituents as adenosine receptor antagonists

Amino-substituted pyrido[2,3-d]pyrimidinediones have previously been found to bind to adenosine A1 and A2A receptors in micromolar concentrations. The present study was aimed at studying the structure-activity relationships of this class of compounds in more detail. Most of the investigated compounds were provided with polar substituents, such as ethoxycarbonyl groups and basic amino functions, in order to improve their water-solubility. The compounds were synthesized starting from 6-amino-1,3-dimethyluracil via different reaction sequences involving (cyano)acetylation, Vilsmeier formylation, or reaction with diethyl ethoxymethylenemalonate (EMME). The most potent and selective compound of the present series was 6-carbethoxy-1,2,3,4-tetrahydro-1,3-dimethyl-5-(2-naphthylmethyl)aminopyrido[2,3-d]pyrimidine-2,4-dione (11c) with a Ki value of 5 nM at rat and 25 nM at human A1 receptors. The compound was more than 60-fold selective versus A3 and more than 300-fold selective versus A2A receptors. It showed an over 300-fold improvement with respect to the lead compound. In GTPgammaS binding studies at membranes of Chinese hamster ovary cells recombinantly expressing the human adenosine A1 receptor, 11c behaved as an antagonist with inverse agonistic activity. A regioisomer of 11c, 6-carbethoxy-1,2,3,4-tetrahydro-1,3-dimethyl-7-(2- naphthylmethyl)aminopyrido[2,3-d]pyrimidine-2,4-dione (7a) in which the 2-naphthylmethylamino substituent at position 5 of 11c was moved to the 7-position, was a relatively potent (Ki=226 nM) and selective (>20-fold) A3 ligand. In the series of compounds lacking an electron-withdrawing ethoxycarbonyl or cyano substituent in the 6-position, compounds with high affinity for adenosine A2A receptors were identified, such as 1,2,3,4-tetrahydro-1,3-dimethyl-5-(1-naphthyl)aminopyrido[2,3-d]pyrimidine-2,4-dione 16b (Ki human A2A=81.3 nM, Ki human A1=153 nM, and Ki human A3>10,000 nM).     

Transversion-specific purine analogue mutagens and the mechanism of hydroxylaminopurine mutagenesis

The tryptophan synthetase gene A series of mutants in E. coli has been used to examine the mutational specificity of over 80 purine base analogues. 4 purine analogues have been discovered that solely cause transversions. Evidence is presented that hydroxylaminopurine mutagenesis is caused by a covalent reaction of these compounds with DNA. The transversion-causing purine analogues are derivatives of 2-aminopurine (2AP) and 2,6-diaminopurine (2,6DAP). They stimulate the full reversion frequency of those trp A which can revert through an AT----CG transversion. 8 purine base analogues have been found that induce the AT----CG transversion at the trp A88 site; and 2-amino-6-methylaminopurine (2A6MAP) stimulates by 124-fold, 2-amino-6-ethylaminopurine by 20-fold, 2-methylaminopurine (2MAP) by 9.4-fold, 2,6-bismethylaminopurine by 25-fold, 2AP by 230-fold, 2,6DAP by 15-fold, 2.6-diaminopurine riboside by 5-fold, and 2-hydroxylaminopurine by 11-fold. The last 4 analogues also cause transitions. 2A6MAP, 2-amino-6-ethylaminopurine and 2,6-bismethylaminopurine stimulate only the AT----CG transversion while 2MAP additionally gives rise to AT----TA transversions. By testing other negative 2AP derivatives, the structural requirements necessary for AT----CG transversion mutagenesis have been determined. All 12 hydroxylaminopurine base analogues tested, 2,6-dimethoxyaminopurine and 2-hydrazinopurine were found to cause transition mutations. All of the compounds stimulated the AT----GC transition (by up to 1000-fold) and 11 of the 14 base analogues raised the GT----AT transition (by up to 450-fold). On increasing the hydroxylaminopurine concentration, the mutation frequency also increased concomitantly. Since 6-hydroxylamino-9-methylpurine and 6-methylhydroxylaminopurine cause transitions, the mechanism of hydroxylaminopurine mutagenesis cannot be entirely due to an alteration in tautomeric equilibria or "wobble" type base mispairing. It is proposed that a major mechanism for hydroxylaminopurine mutagenesis is due to the reaction of these compounds with the O6-position of guanine and the O4-position of thymine.     

Design and synthesis of benzo[c,d]indolone-pyrrolobenzodiazepine conjugates as potential anticancer agents

A series of benzo[c,d]indol-2(1H)one-PBD conjugates (11a-l) have been designed and synthesized as potential anticancer agents. These compounds were prepared by linking the C8-position of DC-81 with a benzo[c,d]indol-2(1H)one moiety through different alkane spacers in good yields and confirmed by (1)H NMR, mass and HRMS data. The DNA binding ability of these conjugates was evaluated by thermal denaturation studies and interestingly, compound 11l showed enhanced DNA binding ability. These compounds were also evaluated for their anticancer activity in selected human cancer cell lines of lung, skin, colon and prostate by using MTT assay method. These new conjugates showed promising anticancer activity with IC(50) values ranging from 1.05 to 36.49 μM. Moreover, cell cycle arrest in SubG1 phase was observed upon treatment of A549 cells with 1 and 2 μM (IC(50)) concentrations of compound 11l and it induced apoptosis. This is confirmed by Annexin V-FITC, Hoechst staining, caspase-3 activity as well as DNA fragmentation analysis.