Novel esters of glaucarubolone as inducers of terminal differentiation of promyelocytic HL-60 cells and inhibitors of 7,12-dimethylbenz[a]anthracene-induced preneoplastic lesion formation in mouse mammary organ culture.

In an effort to discover new chemotherapeutic/chemopreventive agents from natural sources, brusatol (1) was found to induce HL-60 cellular differentiation, accompanied by strong antiproliferative and cytotoxic effects. A series of natural and semisynthetic quassinoids (1-48) was designed to effect both antiproliferative and differentiation-inducing properties. Compounds were assessed in vitro using the HL-60 promyelocytic cell model. Changes in activity due to structural modification of the core structure glaucarubolone (24) were consistent with activities reported in other cell systems. However, the following were novel SAR findings: (1) semisynthetic analogues with a hydroxylated ring at the beta-position of the ester side chain at C-15 were able to induce cellular differentiation at concentrations lower than those inducing cell growth arrest, and (2) quassinoids inhibiting DNA synthesis with greater efficacy than reducing cellular viability possessed alkyl substitutions at the alpha-position of the C-15 ester side chain. Analogues from this latter group and brusatol (1) and bruceantin (2) inhibited dimethylbenz(a)anthracene-induced preneoplastic lesion formation in a mouse mammary organ culture. The novel finding of 1 and glaucarubolone analogues as potent inducers of differentiation leads to potential novel applications in the field of cancer.     

Potential cncer chemopreventive flavonoids from the stems of Tephrosia toxicaria

A new butenylflavanone, (2S)-5-hydroxy-7-methoxy-8-[(E)-3-oxo-1-butenyl]flavanone (1), and a new rotenoid, 4',5'-dihydro-11,5'-dihydroxy-4'-methoxytephrosin (2), as well as three active flavonoids of previously known structure, isoliquiritigenin (3), genistein (4), and chrysoeriol (5), along with nine known inactive compounds, alpha-toxicarol (6), sumatrol, 6a,12a-dehydro-alpha-toxicarol, 11-hydroxytephrosin, obovatin, marmesin, lupenone, benzyl benzoate, and benzyl trans-cinnamate, were isolated from an ethyl acetate-soluble extract of the stems of Tephrosia toxicaria, using a bioassay based on the induction of quinone reductase (QR) in cultured Hepa 1c1c7 mouse hepatoma cells to monitor chromatographic fractionation. The structures of compounds 1 and 2 were elucidated by spectroscopic data interpretation. All isolates were evaluated for their potential cancer chemopreventive properties utilizing an in vitro assay to determine quinone reductase induction. Selected compounds were tested in a mouse mammary organ culture assay to evaluate the inhibition of 7,12-dimethylbenz[a]anthracene (DMBA)-induced preneoplastic lesions.     

Potential cancer chemopreventive constituents of the seeds of Dipteryx odorata (tonka bean)

A new cassane diterpene, dipteryxic acid (1), and a new isoflavonolignan, 5-methoxyxanthocercin A (2), as well as four known active compounds, isoliquiritigenin (3), 6,4'-dihydroxy-3'-methoxyaurone (4), sulfuretin (5), and (+/-)-balanophonin (6), and five known inactive compounds, butin, eriodictyol, 7-hydroxychromone, 7,3'-dihydroxy-8,4'-dimethoxyisoflavone, and (-)-lariciresinol, were isolated from an ethyl acetate-soluble extract of the seeds of Dipteryx odorata, using a bioassay based on the induction of quinone reductase (QR) in cultured Hepa 1c1c7 mouse hepatoma cells to monitor chromatographic fractionation. The structures of compounds 1 and 2 were elucidated by spectroscopic data interpretation. Single-crystal X-ray diffraction analysis was used to confirm the relative stereochemistry of compound 1. Selected compounds (3-5) were evaluated in a mouse mammary organ culture assay, with isoliquiritigenin (3) found to exhibit 76% inhibition at a dose of 10 microg/mL.     

Triterpenoids from the resin of Styrax tonkinensis and their antiproliferative and differentiation effects in human leukemia HL-60 cells

Four new triterpenoids, 6beta-hydroxy-3-oxo-11alpha,12alpha-epoxyolean-28,13beta-olide (1), 3beta,6beta-dihydroxy-11alpha,12alpha-epoxyolean-28,13beta-olide (2), 3beta,6beta-dihydroxy-11-oxo-olean-12-en-28-oic acid (3), and 3beta-hydroxy-12-oxo-13Halpha-olean-28,19beta-olide (4), and five known triterpenes, 19alpha-hydroxy-3-oxo-olean-12-en-28-oic acid (5), 6beta-hydroxy-3-oxo-olean-12-en-28-oic acid (6), sumaresinolic acid (7), siaresinolic acid (8), and oleanolic acid (9), were isolated from the resin of Styrax tonkinensis. The structures of these triterpenoids were determined by physicochemical and spectroscopic methods. The configuration of compound 4 was confirmed by X-ray crystallographic analysis. All these triterpenoids inhibited HL-60 cell growth with IG(50) values ranging from 8.9 to 99.4 microM. Oleanolic acid (9) was the most effective antiproliferative agent, with an IG(50) value of 8.9 microM. While 3beta,6beta-dihydroxy-11-oxo-olean-12-en-28-oic acid (3) exhibited the least effective growth inhibition among these triterpenoids, it induced HL-60 cells to undergo differentiation as measured by an NBT reduction assay.     

Sinulodurins A and B, antiproliferative and anti-invasive diterpenes from the soft coral Sinularia dura

Two new diterpenes, sinulodurin A (1) and sinulodurin B (2), along with two known sterols, 24 S-methyl cholesterol and 24-methylene cholesterol, were isolated from the Palau soft coral Sinularia dura. The structures of the new metabolites were determined on the basis of spectroscopic methods and by comparison of NMR data with those of related metabolites. Sinulodurin A (1) and sinulodurin B (2) showed antiproliferative activity against highly malignant +SA mammary epithelial cells with an IC 50 range of 20-30 microM. They also displayed anti-invasive activity against human highly metastatic prostate cancer PC-3M-CT+ cells in the spheroid disaggregation assay. Furthermore, the antimicrobial activities of the isolates were tested.     

Constituents of the bark and twigs of Artocarpus dadah with cyclooxygenase inhibitory activity

Fractionation of an ethyl acetate-soluble extract of the bark of Artocarpus dadah has led to the isolation of three new prenylated stilbenoid derivatives, 3-(gamma,gamma-dimethylallyl)resveratrol (1), 5-(gamma,gamma-dimethylallyl)oxyresveratrol (2), 3-(2,3-dihydroxy-3-methylbutyl)resveratrol (3), and a new benzofuran derivative, 3-(gamma,gamma-dimethylpropenyl)moracin M (4), along with six known compounds, oxyresveratrol, (+)-catechin, afzelechin-3-O-alpha-L-rhamnopyranoside, (-)-epiafzelechin, dihydromorin, and epiafzelechin-(4beta-->8)-epicatechin. From an ethyl acetate-soluble extract of the twigs of the same plant were isolated compound 4 and two new neolignan derivatives, dadahols A (5) and B (6), as well as 10 known compounds, oxyresveratrol, (+)-catechin, afzelechin-3-O-alpha-L-rhamnopyranoside, resveratrol, steppogenin, moracin M, isogemichalcone B, gemichalcone B, norartocarpetin, and engeletin. The structures of compounds 1-6 were determined using spectroscopic and chemical methods. Isolates were evaluated for their inhibitory effects against both cyclooxygenase-1 (COX-1) and -2 (COX-2) and in a mouse mammary organ culture assay.     

Antitumor agents. 289. Design, synthesis, and anti-breast cancer activity in vivo of 4-amino-2H-benzo[h]chromen-2-one and 4-amino-7,8,9,10-tetrahydro-2H-benzo[h]chromen-2-one analogues with improved water solubility

Previously, we reported that 4-amino-2H-benzo[h]chromen-2-one (ABO) and 4-amino-7,8,9,10-tetrahydro-2H-benzo[h]chromen-2-one (ATBO) analogues, which were developed from the lead natural product neo-tanshinlactone, are potent cytotoxic agents. In order to improve on their water solubility, the diamino analogues and related salts were designed. All synthesized compounds were assayed for cytotoxicity, and selected compounds were evaluated for in vivo anti-mammary epithelial proliferation activity in wild-type mice and mice predisposed for mammary tumors due to Brca1/p53 mutations. The new derivatives 10, 16 (ABO), 22, and 27 (ATBO) were the most active analogues, with IC(50) values of 0.038-0.085 μM in the cytotoxicity assay. Analogue 10 showed around 50-fold improved water solubility compared with the prior lead ABO compound 4-[(4'-methoxyphenyl)amino]-2H-benzo[h]chromen-2-one (3). Compounds 3, 4, 10, and 22 significantly reduced overall numbers of mammary cells, as indicated by the reduction of mammary gland branching in mutant mice. A one-week treatment with 10 resulted in 80% reduction in BrdU-positive cells in the cancer prone mammary gland. These four compounds had differential effects on cellular proliferation and apoptosis in wild-type mouse and a mouse model of human breast cancers. Compound 10 merits further development as a promising anticancer clinical trial candidate.     

Effect of Tangnaikang on TGF-β1-induced transdifferentiation of human renal tubular epithelial HK-2 cells

Objective

To explore the function of Tangnaikang (TNK) in the prevention and treatment of renal interstitial fibrosis through transdifferentiation of the human renal tubular epithelial cell line HK-2 induced by transforming growth factor-β₁ (TGF-β₁).

Methods

HK-2 cells cultured in dulbecco's modified eagle medium/F12 (1:1) with 10% fetal calf serum were divided into six groups: blank control group, TGF-β₁ group (TGF-β₁ 10 ng/mL), serum control group (TGF-β₁ 10 ng/mL + 10% serum), treatment group 1 (TGF-β₁ 10 ng/mL + 5% TNK serum), treatment group 2 (TGF-β₁ 10 ng/mL + 10% TNK serum), and treatment group 3 (TGF-β₁ 10 ng/mL + 20% TNK serum). Cell proliferation was detected by 4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Expression of α-smooth muscle actin (α-SMA) and E-cadherin were observed by immunohistochemical assay. The contents of collagen I (Col I), collagen III (Col III), and fibronectin (FN) in the culture medium supernatant were detected by ELISA.

Results

E-cadherin was expressed and α-SMA was not expressed in normal HK-2 cells. In HK-2 cells cultured with TGF-β₁ α-SMA expression significantly increased, HK-2 cells significantly proliferated, and secretion of Col I, Col III, and FN significantly increased compared with the blank control group (all P<0.05). In the HK-2 cells cultured with TGF-β₁ and TNK serum, the expression of α-SMA significantly decreased, the expression of E-cadherin significantly increased, and the cell proliferation and the secretion of Col I, Col III and FN were significantly inhibited compared with the TGF-β₁ group (all P<0.05).

Conclusion

TNK can inhibit cell proliferation and reduce secretion of Col I, Col III, and FN. This indicates that TNK can inhibit transdifferentiation of human renal tubular epithelial cells induced by TGF-β₁, with the effect of preventing and treating renal interstitial fibrosis.     

miR-223对粒细胞性白血病细胞增殖影响及作用机理

通过在体外将miR-223 duplex转染至粒系白血病细胞株（HL-60、NB4）,观察其对粒系白血病细胞增殖作用的影响,来探讨miR-223对白血病细胞诱导分化及作用机理。以白血病HL-60、NB4细胞株为靶细胞,采用MTT法和半固体集落法观察不同浓度的miR-223对HL-60、NB4细胞增殖的影响;流式细胞术观察粒系细胞分化的表面标志（CD11b＋、CD15＋）表达;RT-PCR检测miR-223对c/EBPα、NFI-A转录因子表达的影响以及用免疫印迹反应观察相应蛋白的表达情况。结果表明：（1）低浓度的miR-223（10nM、20nM）均抑制HL-60、NB4细胞增殖（P〈0.05或P〈0.01）,但在HL-60细胞中随着其浓度的增加（50nM、100nM）,抑制作用与对照组相比无差异;而NB4细胞中随浓度增加抑制作用却没有增强（P〈0.05）。半固体集落培养结果显示与MTT结果相一致。（2）流式细胞术检测用不同浓度的miR-223转染粒系细胞中发现,粒系细胞表面标志CD11b＋表达在低浓度（10nM、20nM）下随其浓度升高而增加,但浓度增至50nM、100nM时表达有所下降;而CD15＋在HL-60细胞中表达无显著差异,NB4细胞中表达趋势与CD11b＋一致。（3）两细胞在miR-223作用前后提取总RNA分别进行RT-PCR检测显示,NB4细胞中NFI-A转录因子表达随浓度增加而降低,分别低于未经处理的0.6倍、2.0倍、1.1倍和1.5倍。c/EBPα基因表达随浓度增加而增加,分别是未经处理细胞的1.5倍、2.0倍、1.8倍和2.0倍。而在HL-60细胞中c/EBPα基因表达无明显差异,NFI-A仅在20nM浓度下表达下降差异显著,低于未经处理细胞的3.0倍。对NB4细胞中的NFI-A蛋白免疫印迹分析发现,经miR-223处理过的细胞中NFI-A蛋白含量随浓度增加而有所下降。结论：转染一定浓度miR-223至粒系白血病细胞中,能够抑制恶性细胞克隆增殖,其作用机制有可能通过竞争性抑制NFI-A转录因子表达,恢复与分化相关的c/EBPα转录因子的表达,来诱导粒系白血病细胞向成熟方向分化。     

In vitro antiproliferative and antioxidant activities and total phenolic contents of the extracts of Melastoma malabathricum leaves

The present study aims to determine the in vitro antiproliferative and antioxidant activities of various extracts from the leaves of Melastoma malabathricum using various established in vitro assays. The aqueous extract inhibited the proliferation of Caov-3 and HL-60 cell lines, while the chloroform extract exhibited antiproliferative activity against the Caov-3, HL-60, and CEM-SS cell lines. The methanol extract demonstrated antiproliferative activity against more cell lines, including the MCF-7, HeLa, Caov-3, HL-60, CEM-SS, and MDA-MB-231 cancer cell lines. Interestingly, all extracts did not inhibit the proliferation of 3T3 cells, thus indicating their noncytotoxic properties. Unlike the chloroform extracts, the aqueous and methanol extracts of M malabathricum (20, 100, and 500 μg/ml) produced high antioxidant activity for the superoxide scavenging assay with only the 500 μg/ml aqueous and methanol extracts exhibited high activity for the 2,2-diphenyl -1-picrylhydrazyl radical scavenging assay. The total phenolic content recorded for the aqueous, methanol, and chloroform extracts were 3344.2 ± 19.1, 3055.1 ± 8.7, and 92.5 ± 7.3 mg/100 g of gallic acid, respectively. The M malabathricum leaves possessed potential antiproliferative and antioxidant activities that could be attributed to its high content of phenolic compounds.     

ent-Labdane diterpenoid lactone stereoisomers from Andrographis paniculata

Two pairs of ent-labdane diterpenoid lactone stereoisomers (1- 4) including three new compounds (1- 3) were isolated from the 85% EtOH extract of the aerial parts of Andrographis paniculata. The structures of these compounds were identified as 7 R-hydroxy-14-deoxyandrographolide (1), 7 S-hydroxy-14-deoxyandrographolide (2), 12 S,13 S-hydroxyandrographolide (3), and 12 R,13 R-hydroxyandrographolide (4) by spectroscopic data analyses and calculated (13)C NMR data at the B3LYP/6-311++G(2d,p)//B3LYP/6-31G* level using the GIAO method. The 12 S-configuration of 4 was revised to 12 R based on the spectroscopic data. The antiproliferative activities of the two pairs of stereoisomers and 14 other ent-labdane diterpenoid derivatives were determined in human leukemia HL-60 cells. Andrographolide (7) and isoandrographolide (12) exhibited higher antiproliferative activities than other ent-labdane diterpenoids with GI 50's of 9.33 and 6.30 microM, respectively.     

Constituents of the stem bark of Pongamia pinnata with the potential to induce quinone reductase

Activity-guided fractionation of the petroleum ether and ethyl acetate extracts of the stem bark of Pongamia pinnata, using cultured Hepa 1c1c7 mouse hepatoma cells to evaluate quinone reductase (QR) inducing activity, led to the isolation of four new flavanone derivatives (1-4), one new flavone (5), one new chalcone (6), and 13 known compounds of the flavonoid, terpenoid, and fatty acid types. The structures of 1-6 were characterized on the basis of the interpretation of their spectroscopic data. The absolute stereochemistry of compounds 1-4 was determined from their CD data and by Mosher ester determination. All isolates obtained were evaluated in the quinone reductase induction assay.     

力达霉素抑制HL-60细胞增殖和诱导分化作用

 目的 研究力达霉素对人急性髓性白血病细胞(HL-60) 增殖和诱导分化的作用。方法 用不同浓度的力达霉素处理HL-60 细胞,通过MTT法检测力达霉素对细胞的增殖抑制作用;利用瑞氏-姬姆萨染色法观察力达霉素对细胞形态的影响;通过硝基蓝四氮(NBT)还原比色法测定力达霉素对HL-60细胞的诱导分化作用。结果 不同浓度力达霉素处理细胞后,HL-60 细胞的增殖能力明显受到抑制。当力达霉素的作用浓度超过100 μmol·L-1时,抑制率可高达99%,并且抑制作用呈现时间依赖性和剂量依赖性的特点。Wright-Giemsa染色结果显示,力达霉素作用HL-60 细胞后,细胞在形态学方面发生很大变化。同时,力达霉素能够提高细胞的NBT还原能力。1 nmol·L-1和0.1 nmol·L-1浓度的力达霉素可以使HL-60细胞的NBT还原率分别提高到39.2% 和54.5 % ;0.01 nmol·L-1和0.001 nmol·L-1浓度的力达霉素则使阳性细胞的百分率增加到78.6%和89.9% ,与对照组相比,差异具有显著性（P<0.01）。结论 力达霉素具有较强的抑制HL-60细胞增殖的能力,同时,低浓度力达霉素能使HL-60细胞形态发生改变,NBT还原阳性率升高,诱导细胞发生分化,其作用机制有待于进一步深入研究。     

Three new C-14 oxygenated taxanes from the wood of Taxus yunnanensis

Three new C-14 oxygenated taxane-type diterpenes, hongdoushans A-C (1-3), were isolated from the wood of Taxus yunnanensis together with four known diterpenes and two lignans. The absolute stereochemistry of the 2-methylbutyryloxy group attached at C-14 of the taxane skeleton was determined to be S by GC analysis of the methyl ester of 2-methylbutyric acid obtained after alkaline hydrolysis of 1 and 4 followed by treatment with CH(2)N(2). The complete stereostructure of the known compound 2alpha,5alpha,10beta-triacetoxy-14beta-[(S)-2-methylbutyryloxy]-4(20),11-taxadiene (4) was established for the first time. The isolates obtained were evaluated for their antiproliferative activity toward murine colon 26-L5 carcinoma and human HT-1080 fibrosarcoma cell lines.     

小鼠阳和汤血清对细胞的诱导分化作用

目的观察小鼠阳和汤血清对细胞的诱导分化作用.方法SRB法和乳酸脱氢酶释放法检测B16细胞活性;油红染色法检测3T3-L1前脂肪细胞的分化;NBT还原能力和细胞吞噬功能法检测HL-60细胞的分化;血红蛋白含量法检测K562细胞的分化;黑色素含量法检测B16细胞的分化.结果小鼠阳和汤血清能明显呈剂量依赖性抑制B16细胞生长,但不表现细胞毒作用;阳和汤血清能促进3T3-L1前脂肪细胞在分化液刺激下的油红染色,提高HL-60细胞的NBT还原能力和对辣根过氧化物酶的吞噬功能,增加K562细胞的血红蛋白含量,降低B16细胞的黑色素含量.结论小鼠阳和汤血清对3T3-L1前脂肪细胞、肿瘤细胞均有诱导分化和成熟作用,这可能是其抗肿瘤作用的主要机制.     

中药血清对大鼠bMSCs体外成软骨分化中Col2a1和Acan表达的影响

目的观察中药雪莲花、丹参、仙灵脾、黄芪和复方右归饮含药血清对大鼠骨髓间充质干细胞(bMSCs)成软骨分化中Ⅱ型胶原α1(Col2a1)和软骨聚糖蛋白(Acan)表达的影响。方法 2月龄SD大鼠分离骨髓间充质干细胞,置含15%胎牛血清的DMEM/F12(1∶1)培养基中培养。传至第3代后,将培养细胞分为未诱导组、TGF诱导组、中药血清诱导组和TGF-中药血清联合诱导组。中药血清的使用浓度为5%,由上述中药连续大鼠灌胃3 d后,分离外周血获得。各组细胞诱导至7 d和14 d时,分别提取总RNA,逆转录成cDNA,荧光定量PCR法检测Col2a1和Acan的表达,同时应用细胞免疫化学法检测诱导14 d的Ⅱ型胶原的表达。结果 bMSCs在TGF-β1的诱导下可以分化为软骨样细胞,在诱导后7 d和14 d,TGF诱导组Col2a1的表达量显著高于未诱导组,分别为14.272倍和35.833倍,中药血清组Col2a1的表达量变化不显著,TGF-中药血清联合诱导组Col2a1的表达量虽高于未诱导组,但显著低于TGF诱导组。Acan基因的表达量在各组中均显著低于未诱导组。结论骨髓间充质干细胞在体外可以分化为软骨样细胞,中药对细胞成软骨分化没有明显的促进作用,并对TGF诱导效应具有一定的抑制作用。     

Chemical and biological investigation of the fungus Pulveroboletus ravenelii

Two new compounds, pulveraven A (1) and pulveraven B (2), as well as vulpinic acid (3) and its previously unreported polymorph were isolated from the fruiting body of Pulveroboletus ravenelii. The structures were determined using a combination of NMR, MS, IR, optical rotation, molecular modeling, and X-ray analysis. The isolates were evaluated for antimicrobial activity as well as their potential to inhibit cyclooxygenase (COX) activity and carcinogen-induced preneoplastic lesion formation with mouse mammary organ culture (MMOC).     

羊栖菜多糖诱导肿瘤细胞凋亡的实验研究

目的研究羊栖菜多糖(SFPS)对人白血病HL-60细胞系增殖抑制和凋亡诱导作用。方法MTT法检测SFPS对HL-60细胞增殖的抑制作用;扫描电镜、透射电镜、琼脂糖凝胶电泳和流式细胞仪检测细胞凋亡。结果SFPS对HL-60细胞具有显著生长抑制作用,并呈量效和时效关系;药物浓度为300 mg/L和500 mg/L作用HL-60细胞后,观察到典型的细胞凋亡形态学特征;DNA凝胶电泳呈现梯状条带;DNA直方图出现亚G1峰。在一定浓度范围内,SFPS诱导细胞凋亡的作用呈现浓度和时间依赖性,同时G2/M期细胞比例增多。结论SFPS抗肿瘤作用与诱导细胞凋亡和G2/M期细胞阻滞有关。     

SPGL诱导HL-60细胞分化作用的研究

目的:研究光叶海桐总皂苷(SPGL)诱导HL-60细胞分化的作用。方法:采用1×10-5~5×10-4mg/ml浓度SPGL处理第3 d的HL-60细胞进行Wrigh-Giemsa染色、NBT还原能力及细胞表面抗原的检测。结果:在药物作用下,HL-60细胞形态学出现分化特征;NBT还原能力增强,且其阳性细胞数与SPGL的浓度有明显的量-效关系;CD11b与CD14表面抗原的表达明显升高。结论:SPGL有诱导HL-60细胞分化的作用,并诱导其向粒细胞系和单核系细胞分化。     

Isolation and characterization of miscellaneous secondary metabolites of Deprea subtriflora

Two new C-18 norwithanolides based on a C(27) skeleton, subtrifloralactones K (1) and L (2), a new C-18 oxygenated withanolide, 13 beta-hydroxymethylsubtrifloralactone E (3), and a new alpha-ionone derivative, (+)-7 alpha,8 alpha-epoxyblumenol B (4), along with five known compounds, philadelphicalactone A (5), (2S,3S,4R)-2-[(2R)-2'-hydroxytetracosanoylamino]-1,3,4-octadecanetriol (6), trans-N-feruloyltyramine, cis-N-feruloyltyramine, and (S)-coriolic acid, were isolated from additional active fractions of the chloroform-soluble extract of Deprea subtriflora, using a quinone reductase (QR) induction assay as a monitor. The structures of compounds 1-4 were characterized by spectroscopic data interpretation. The potential cancer chemopreventive activities of all isolates in terms of their ability to induce QR activity with cultured Hepa 1c1c7 mouse hepatoma cells were evaluated.