黄樟素氧化物对去除成纤维细胞生长因子所诱导的血管内皮细胞凋亡及生长的影响

目的:研究黄樟素氧化物对去除成纤维细胞生长因子(FGF)诱导的血管内皮细胞凋亡及生长的影响.方法:光学显微镜观察细胞形态学变化;MTT法测定细胞生长;DNA电泳和荧光显微技术检测DNA断裂;流式细胞术测定细胞周期分布.结果:黄樟素氧化物5-25mg/L处理去除FGF的血管内皮细胞24h,细胞铺展和生长被促进,细胞脱壁和DNA片段化被抑制,黄樟素氧化物10mg/L对细胞周期分布无明显影响.黄樟素氧化物50-100 mg/L促进血管内皮细胞的脱壁和DNA片段化,黄樟素氧化物100mg/L将细胞周期阻断于G_2-M期.结论:黄樟素氧化物在10mg/L时抑制血管内皮细胞凋亡,而在100mg/L时促进其凋亡,该药物对血管内皮细胞生长和凋亡有重要影响.     

Safrole oxide inhibits angiogenesis by inducing apoptosis

Our previous studies indicate that 3, 4-(methylenedioxy)-1-(2', 3'-epoxypropyl)-benzene (safrole oxide), a newly synthesized compound, induces apoptosis in vascular endothelial cells (VECs) and A549 lung cancer cells. To our knowledge, the inhibition of angiogenesis by safrole oxide has not been reported yet. We report here that cultured rat aorta treated with safrole oxide exhibited a significant microvessel reduction as determined by counting the number of microvessels in a phase contrast microscope. There were more microvessels formed in the presence of A549 lung cancer cells in rat aorta model, while a dramatic inhibition of angiogenesis was obtained by adding 220-450 micromol l(-1) of safrole oxide to the growth medium (P<.01). The culture of rat aorta treated with safrole oxide produced only some abortive endothelial cells but not microvessels. Furthermore, safrole oxide induced antiangiogenic effect in the chorioallantoic membranes (CAM) as a dose dependent manner. Eggs treated with 2-11 micromol 100 microl(-1) per egg of the safrole oxide for 48 h exhibited a significant reduction in blood vessel area of the CAM, a process likely mediated by apoptosis as demonstrated by DNA fragmentation. Our results suggest that safrole oxide has antiangiogenic activity and this effect might occur by induction of cellular apoptosis.     

Stimulus effects of N-monoethyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (MDE) and N-hydroxy-1-(3,4-methylenedioxyphenyl)-2-aminopropane (N-OH MDA) in rats trained to discriminate MDMA from saline

Tests of stimulus generalization were conducted using rats trained to discriminate 1.5 mg/kg of N-monomethyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane HCl (MDMA) from saline in order to determine if two structurally related analogs (MDE and N-OH MDA) would produce similar stimulus effects. The MDMA-stimulus (MDMA, ED50 value = 0.76 mg/kg) generalized both to MDE (ED50 value = 0.73 mg/kg) and N-OH MDA (ED50 value = 0.47 mg/kg). Administration of (+)amphetamine resulted in partial generalization (maximum of 49% MDMA-appropriate responding) in the MDMA-trained animals. Taken together with our previous studies showing that MDMA substitutes for the phenylisopropylamine stimulant (+)amphetamine, but that neither MDE nor N-OH MDA substitute for (+)amphetamine or for the phenylisopropylamine hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM), the present results [i.e., MDMA-stimulus generalization to MDE, N-OH MDA, but not to (+)amphetamine] suggest that 1) MDMA produces effects other than those that may be considered amphetamine-like, and 2) MDE and N-OH MDA are MDMA-like agents with even less of an amphetamine-like component of action than MDMA itself.     

S(+)- and R(-)N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA) as discriminative stimuli: effect of cocaine

Racemic N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (methylenedioxymethamphetamine, MDMA), a central stimulant and empathogenic agent, and cocaine are drugs of abuse that function as training drugs in drug discrimination studies. In tests of stimulus generalization (substitution), asymmetric generalization occurs between the two agents: a (+/-)MDMA stimulus generalized to cocaine, but a cocaine stimulus did not generalize to (+/-)MDMA. A possible explanation may be found, at least in part, in the stimulus effects of the optical isomers of MDMA. In the present study, groups of male Sprague-Dawley rats were trained to discriminate either S(+)MDMA (training dose=1.5 mg/kg, i.p.; n=10; ED50=0.6 mg/kg) or R(-)MDMA (training dose=1.75 mg/kg, i.p.; n=7; ED50=0.4 mg/kg) from saline vehicle using a VI-15s schedule of reinforcement. Tests of stimulus generalization with cocaine were conducted in each of the two groups. Cocaine only partially substituted for the S(+)MDMA stimulus (maximum=39% drug-appropriate responding), and various doses of cocaine did not enhance the percent drug-appropriate responding produced by a low dose (0.5 mg/kg) of S(+)MDMA. In contrast, the R(-)MDMA stimulus generalized completely to cocaine (ED50=1.3 mg/kg). Taken together with an earlier report that a (+/-)MDMA stimulus generalizes to cocaine, it would seem that the stimulus actions of cocaine might share greater similarity with R(-)MDMA than with S(+)MDMA.     

Redifferentiation of human hepatoma cell induced by 6-(p-chlorophenyl)-3-[1-(p-chlorophenyl)-5-methyl-1 H-1,2,3-triazol-4-yl]-s-triazolo[3,4-b]-1,3,4-thiadiazole (TDZ)

6-(p-Chlorophenyl)-3-[1-(p-chlorophenyl)-5-methyl-1 H-1,2,3-triazol-4-yl]-s-triazolo[3,4-b]-1,3,4-thiadiazole (TDZ) is a derivative of various substituted s-triazolo[3,4-b]-1,3,4-thiadiazoles, which are associated with diverse pharmacological activities. However, the antitumor activity of TDZ is not well understood. To evaluate its role on tumor cell lines, we have examined the effect of TDZ on two tumor lines: human hepatoma cell (SMMC-7721) in vitro and Sarcoma180 tumor (S180) in vivo. The cytotoxicity of TDZ on human hepatoma cells was assessed using the MTT assay. The inhibition on tumor growth was evaluated by means of trypan blue exclusion test in vitro, and using a Sarcoma180 tumor (S180) animal model in vivo. A scanning electronic microscope was used to discover the morphological changes on cell surface, cell electrophoresis was employed to determine the changes of cell surface negative charges, and alpha-fetoprotein was applied as a biomarker of hepatoma. The effect of TDZ on DNA synthesis was determined by a [3H]-thymidine incorporation assay, and cell cycle distribution by flow cytometry. The IC50 value of TDZ on SMMC-7721 cells was 52.9 microg/ml (48 h). However, TDZ could inhibit the growth of SMMC-7721 cells at concentrations far lower than the IC50 value. Treated with the same low concentrations of TDZ, microvilli on the surface of SMMC-7721 cells decreased obviously, electrophoresis rate of cells reduced from 2.14 microm ms(-1) x V(-1) x cm(-1) of control to 1.54 and 1.56 microm x s(-1) x V-1 x cm(-1), the content of AFP dropped from 205.14 +/- 6.41 ng x mg(-1) Pr to 115.68 +/- 3.47 and 78.57 +/- 2.35 ng mg(-1) Pr, and the DNA replication was inhibited by 26.8% and 45.2%. These results indicated that TDZ may inhibit proliferation of cancer cells by reversing SMMC-7721 cells malignant phenotypic characteristics and inducing redifferentiation. Flow cytometry showed that TDZ-treated cells resulted in a higher proportion of cells in S phase compared with untreated cells, and only when the concentration reached 64 microg/ml, the apoptosis could happen at the rate of 4.2%. Detection of the inhibition of Sarcoma 180 tumor growth in vivo showed that TDZ reduced the tumor weight and 69.08% of the growth was inhibited. TDZ could inhibit the proliferation of tumors in vitro and in vivo; the possible antitumor mechanism might be inducing redifferentiation at a lower dosage on vitro.     

N-Methyl-1-(4-methoxyphenyl)-2-aminopropane (PMMA) and N-Methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA) produce non-identical discriminative stimuli in rats

N-Methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (methylenedioxymethamphetamine, MDMA, Ecstasy) and its structurally abbreviated congener N-methyl-1-(4-methoxyphenyl)-2-aminopropane (para-methoxymethamphetamine, PMMA) are chemically related designer drugs, and PMMA is sometimes sold on the clandestine market as a substitute for MDMA. Prior drug discrimination studies have found that MDMA and PMMA substitute for one another suggesting that they produce similar discriminative stimulus effects in rats. However, there also are some indications that the two agents produce distinct stimulus effects. In this study, further comparisons were made between the stimulus effects of these two agents. Sprague-Dawley rats were trained to discriminate either 1.25 mg/kg of PMMA or 1.5 mg/kg of MDMA from saline vehicle in a two-lever operant paradigm. A structure-activity comparison revealed that MDMA and PMMA behave similarly upon homologation of their terminal amine substituents. In contrast, the PMMA stimulus, unlike an MDMA stimulus, failed to generalize completely to the psychostimulant cocaine, 8-hydroxy-2-(N,N-di-n-propylamino)tetralin (8-OH DPAT), and R(-)-1-(3-methoxyphenyl)-2-aminopropane [R(-)MMA]. In an additional group of animals, a (+)amphetamine stimulus partially generalized to R(-)MMA. Taken together, the results argue and re-emphasize the conclusion that the stimulus effects produced by MDMA and PMMA are similar, but non-identical, and that PMMA is the less "stimulant-like" of the two.     

Further studies on N-methyl-1(3,4-methylenedioxyphenyl)-2-aminopropane as a discriminative stimulus: antagonism by 5-hydroxytryptamine3 antagonists.

Using a standard two-lever operant paradigm, male Sprague-Dawley rats were trained to discriminate 1.5 mg/kg N-methyl-1(3,4-methylenedioxyphenyl)-2- aminopropane (MDMA) from saline using a variable-interval 15-s schedule of reinforcement for food reward. Tests of stimulus antagonism were conducted to further define the mechanism of action of MDMA as a discriminative stimulus. Low doses of the 5-hydroxytryptamine1A (5-HT1A) antagonist NAN-190, the 5-HT2 antagonist pirenperone, and the dopamine antagonist haloperidol were able to somewhat attenuate the MDMA stimulus; however, none of these agents decreased MDMA-appropriate responding to less than 46%. The 5-HT3 antagonists zacopride and LY 278584 (ID50 = 0.02 micrograms/kg) antagonized the MDMA discriminative stimulus. Zacopride also attenuated the stimulus effects of 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) in DOM-trained animals but not those of (+)amphetamine in (+)amphetamine-trained animals. Several possible mechanistic interpretations are provided but it is concluded that MDMA produces its stimulus effects via a complex mechanism involving both dopaminergic and serotonergic components.     

Notch1(NICD)过表达真核载体构建及对大鼠BMSCs增殖分化的影响

目的 构建Notch1(NICD)过表达真核载体,探讨其对大鼠骨髓间充质干细胞（bone mesenehymal stem cells,BMSCs）增殖分化的影响。方法 以大鼠BMSCs为研究对象,构建Notch1(NICD)过表达真核载体,将pEGFP-N1-NICD质粒转染BMSCs,实验分为空白对照CON组、空载体组和转染组,转染48h后观察细胞一般形态,Realtime PCR和Western blotting检测NSE、GFAP 和Notch1基因和蛋白表达,流式检测转染后细胞凋亡和细胞周期情况;MTT检测增殖情况。结果 经DNA测序结果,重组质粒pEGFP-N1-NICD编码序列与设计完全一致,转染48h后转染组和空载体组的BMSCs均可表达绿色荧光。转染组Notch1、GFAP基因相对表达量显著高于空载体组和CON组（P＜0.05）,Western blotting检测结果与之类似。转染48h后,转染组活细胞比率及早期凋亡率、晚期凋亡率与CON组和空载体组比较差异均具有统计学意义（P＜0.05）;空载体组与CON组晚期凋亡率比较差异具有统计学意义（P＜0.05）,转染组细胞处于G1/G0细胞比例显著高于CON组和空载体组（P＜0.01）,而处于S和G2/M期细胞比例显著低于CON组和空载体组（P＜0.01）,转染组增殖曲线值随时间逐渐低于CON组和空载体组,到第4d时显著低于CON组和空载体组（P＜0.05）。结论Notch1(NICD)过表达真核载体构建成功,高表达Notch1(NICD)基因可能在一定程度上诱导BMSCs凋亡、抑制其增殖,且表现诱导向神经胶质样细胞分化。     

对映体选择性测定尿中N-甲基-1-(3,4-亚甲二氧苯基)-2-丙胺及其代谢物

建立了尿中Ｎ－甲基－（３，４－亚甲二氧苯基）－２－丙胺及其代谢物对映体浓度的分析方法。药物经手性衍生化后，于非手性ＧＣ固定相上分离。采用该法测定了大鼠屎中各对映体浓度，结果表明药物及其代谢物均呈立体选择性代谢．     

Expression of 14-3-3delta, cdc2 and cyclin B proteins related to exotoxin A-induced apoptosis in HeLa S3 cells

After reports on regression of cancer in humans and animals infected with microbial pathogens date back more than 100 years, much effort has been spent over the years in developing wild type or attenuated bacterial and purified bacterial proteins for the treatment of cancer. Pseudomonas aeruginosa exotoxin A (ETA) is known to inhibit cell growth and trigger significant cell death in various cancer cells. Although ETA induces apoptosis of cancer cells, its exact mechanism of action is not yet known. Four different assays were performed in this study: morphological assessment of apoptotic cells, cell cytotoxicity, cell cycle analysis and Western blot analysis. The proliferation and survival in the cells treated with ETA was decreased. In addition, percentages of apoptotic HeLa S(3) cells treated with ETA were increased. ETA-induced apoptosis rates were confirmed to have increased in a dose-dependent manner through annexin V binding assay. Flow cytometric analysis was examined to ascertain whether ETA could regulate cell cycle in HeLa S(3) cells. ETA treatment demonstrated that the expression of 14-3-3delta proteins was increased, while expression of cdc and cyclin B proteins was decreased, suggesting that ETA induces cell cycle arrest and then progresses to apoptosis. Therefore, these results suggest that P. aeruginosa ETA induced apoptosis in HeLa S(3) cells.     

1-氨基-2-丙醇的拆分

(2S,3S)-(-)-酒石酸于水中拆分消旋1-氨基-2-丙醇,然后用强酸性阳离子树脂分离,得到核苷类药物合成中间体(R)-(-)-1-氨基-2-丙醇,收率66.6%,拆分剂可直接回收利用.     

Derivatives of (R,S)-1-N-(theophyllinyl-7'-ethyl)-amino-2-propanol with antiarrhythmic activity

Three (R,S)-1-N-(theophyllinyl-7'-ethyl)-amino-2-propanol derivatives were obtained as soluble hydrochlorides and tested in four models of experimental arrhythmia. Two compounds 1a and 2 have shown antiarrhythmic properties and very low toxicity.     

Effect of 1-(3,4-methylenedioxyphenyl)-2-aminopropane and its optical isomers in PMMA-trained rats

1-(3,4-Methylenedioxyphenyl)-2-aminopropane (MDA) is a drug of abuse that is known to produce stimulus effects similar to those of the stimulant phenylalkylamine (+)amphetamine and the hallucinogenic phenylalkylamine 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). Earlier, a working model was described to account for the stimulus effects produced by phenylalkylamines. Such agents can produce one or more of three distinct effects: an amphetamine effect, a DOM effect and a third effect that is typified by the agent N-methyl-1-(4-methoxyphenyl)-2-aminopropane (PMMA). Because MDA is known to produce two of the three effects, in the present investigation, we sought to determine if racemic MDA or either of its optical isomers could produce a PMMA-like effect in animals. Administration of S(+)MDA, R(-)MDA and (+/-)MDA to rats trained to discriminate 1.25 mg/kg of PMMA from saline vehicle under a VI 15-s schedule of reinforcement resulted in substitution in each case. (+/-)MDA and S(+)MDA were nearly equipotent and several fold more potent than R(-)MDA. The results are not only consistent with the proposed model but also identify (+/-)MDA as the first phenylalkylamine shown to produce all three types of stimulus effects (i.e., amphetamine-like, DOM-like and PMMA-like) in rats.     

(S)-(+)-2-氨基丙醇的KBH4还原合成法

(S) - ( + ) - 2 -氨基丙醇 ( 1 )为合成左氟沙星的关键手性中间体。本刊曾发表用 Li Al H4 为还原剂 ,把L-氨基丙酸 ( 2 )直接还原制得 1的工艺 ,及用Na BH4 或 KBH4 的 BER树脂为还原剂还原 L-氨基丙酸乙酯盐酸盐 ( 3a)来制备 1的方法 [1～ 4 ]。文献 [5]也报道了用高沸点的乙二醇二甲醚为溶剂 ,把KBH4 和 Zn Cl2 制成 Zn( BH4 ) 2 还原剂 ,还原 3a制备 1来提高收率 ( 60 % )的还原方法。除文献[1] 外 ,其它文献均从 2的乙酯盐酸盐 ( 3a)用不同的还原剂还原制得 1。还原剂 Li Al H4 和 Na BH4 价格较昂贵 ,不宜采用。KBH4 的…     

1,25-二羟维生素D_3对人胃腺癌细胞诱导分化的实验研究

目的观察1,25-二羟维生素D3对胃腺癌MGC-803细胞的诱导分化作用。方法1,25-二羟维生素D3(10-6、10-5、10-4mmol.L-1)作用于MGC-803细胞后,应用四甲基偶氮唑蓝(MTT)法测定细胞增殖能力、平板克隆试验测定细胞集落形成率、流式细胞仪测定细胞周期、TRAP-银染法测定端粒酶活性。结果1,25-二羟维生素D3作用24、48、72和96h后胃腺癌细胞生长均明显受抑制,48h后细胞周期出现向G0/G1期移行的特征性动力学改变,端粒酶活性明显受到抑制。细胞集落形成率明显下降(P<0.05)。结论1,25-二羟维生素D3对人胃腺癌细胞有诱导分化作用。     

Ligustrazine inhibits lipopolysaccharide-induced proliferation by affecting P27, Bcl-2 expression in rat mesangial cells

Ligustrazine has a renoprotective effect against nephritis. In the present study, we investigated the roles of ligustrazine on lipopolysaccharide-induced changes of proliferation, cell cycle in cultured rat mesangial cells. 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay revealed that rat mesangial cells treated with lipopolysaccharide (10mg/l) underwent significant proliferation compared with control group. This effect was significantly inhibited by ligustrazine (400 to 2500 mg/l). Flow cytometric analysis revealed that cells treated with lipopolysaccharide showed significant reduction in the ratio of G0/G1 phase and significant elevation in the ratio of S+G2/M phase. The changes of cell cycle induced by lipopolysaccharide were reversed by ligustrazine. In addition, lipopolysaccharide suppressed P27 protein expression was significantly increased by ligustrazine (100, 500, 2500 mg/l). Moreover, rat mesangial cells treated with lipopolysaccharide showed scanty apoptosis with up-regulation of Bcl-2expression, while Bax protein expression was not changed. Ligustrazine (100, 500, 2500 mg/l) significantly reversed lipopolysaccharide-induced up-regulation of Bcl-2 protein and increased apoptotic cell death. In summary, ligustrazine displayed a significant inhibiting effect on lipopolysaccharide-induced proliferation through increasing P27 and decreasing Bcl-2 protein expression in rat mesangial cells.     

4-[4″-(2″,2″,6″,6″-四甲基-1″-哌啶氮氧自由基)氨基]-4′-去甲表鬼臼毒素对K562/ADM细胞的抑制作用

目的比较4-[4″-(2″,2″,6″,6″-四甲基-1″-哌啶氮氧自由基)氨基]-4′-去甲表鬼臼毒素(GP-7)对多药耐药人慢性粒细胞白血病K562的多柔比星耐药株细胞(K562/ADM细胞)的抑制作用是否优于依托泊苷。方法以依托泊苷和K562细胞为对照,用不同浓度GP-7处理K562/ADM细胞不同时间,MTT比色法测定细胞增殖,流式细胞仪测定细胞周期和细胞凋亡率,普通光学显微镜观察细胞凋亡形态,琼脂糖凝胶电泳观察细胞DNA凋亡性降解。结果8~128mol.L-1GP-7处理48h或64μmol.L-1GP-7处理24~72h,GP-7对K562/ADM细胞的增殖抑制呈剂量依赖性(r=0.947,P<0.05)和时间依赖性(r=0.999,P<0.01)。GP-7及依托泊苷对K562/ADM的IC50分别为(45.9±1.8)及(68.7±4.6)μmol.L-1;64μmol.L-1GP-7作用48h可使G2/M期细胞明显增多,相同情况下依托泊苷则使S期细胞明显增多;GP-7可引起K562/ADM和K562细胞凋亡,但其引起的K562/ADM和K562细胞凋亡率与依托泊苷无明显差异;GP-7可引起K562/ADM和K562细胞典型的凋亡形态学变化和DNA凋亡性降解,但GP-7引起的K562/ADM细胞DNA凋亡性降解弱于K562细胞;128及256μmol.L-1GP-7或依托泊苷处理K562/ADM和K562细胞48h,GP-7诱导DNA凋亡性降解的作用强于依托泊苷,但32和64μmol.L-1时作用则相反。结论GP-7可抑制多药耐药白血病细胞株K562/ADM的增殖,诱导细胞凋亡。GP-7抑制多药耐药白血病细胞株K562/ADM的作用优于依托泊苷。     

1,25-二羟基维生素D_3抑制K562细胞增殖及其机制

目的观察1,25二-羟基维生素D3〔1,25(OH)2D3〕对白血病细胞株K562增殖的影响并初步探讨其作用机制。方法间接免疫荧光法鉴定维生素D受体(VDR)在K562细胞的表达;四噻唑蓝法(MTT)、AO/EB、流式细胞PI单染分析细胞生长抑制率、细胞凋亡率及细胞周期;比色法检测凋亡信号蛋白———天冬酰胺特异酶切的半胱氨酸蛋白酶-3(Caspase-3)活性。结果①K562细胞核阳性表达VDR;②10-8mol.L-11,25(OH)2D3可明显抑制K562细胞增殖,促进凋亡,凋亡率从4.1%(对照组)增至26.5%,P<0.01;③1,25(OH)2D3作用后K562细胞的Caspase-3活性增加,P<0.05,提示细胞凋亡伴随着Caspaes-3活性升高。结论1,25(OH)2D3可明显抑制K562细胞增殖,促进细胞凋亡,其作用机制与Caspase-3活性增加相关。     

Synthesis, characterization, antioxidant, and anticancer studies of 6-[3-(4-chlorophenyl)-1H-pyrazol-4-yl]-3-[(2-naphthyloxy)methyl][1,2,4]triazolo[3,4-b][1,3,4]thiadiazole in HepG2 cell lines

Triazolo- thiadiazoles exhibit a variety of pharmacological properties, due to their cytotoxicity. In continuation of a previous study on triazolo-thiadiazoles, the authors have synthesized a new thiadiazole, 6-[3-(4-chlorophenyl)-1-H-pyrazol-4-yl]-3-[(2-naphthyloxy)methyl][1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (CPNT), which was further characterized by advanced spectral techniques and elemental analysis. The compound exhibited a dose-dependent cytotoxic effect on hepatocellular carcinoma cell line, HepG2 with very low IC₅₀ value of 0.8 μg/ml in 24 h when compared with standard drug, doxorubicin. Incorporation of [³H] thymidine in conjunction with cell cycle analysis suggested that CPNT inhibited the growth of HepG2 cells. Flow cytometric studies revealed more percentage of cells in subG1 phase, indicating apoptosis, which was further confirmed through chromatin condensation studies by Hoechst staining. In vitro antioxidant activity of CPNT was determined by DPPH and ABTS free radical scavenging assays which revealed increasing scavenging activity with increasing concentration of the compound when compared with reference ascorbic acid.     

Comparison of the efficacy of 7-hydroxystaurosporine (UCN-01) and other staurosporine analogs to abrogate cisplatin-induced cell cycle arrest in human breast cancer cell lines

DNA-damaging agents such as cisplatin arrest cell cycle progression at either the G1, S, or G2 phase, although the G1 arrest is seen only in cells expressing the wild-type p53 tumor suppressor protein. Caffeine has been shown to abrogate the S and G2 arrest in p53-defective cells and to enhance cytotoxicity, but at concentrations too toxic to administer to humans. We have reported that 7-hydroxystaurosporine (UCN-01) also overcomes S and G2 phase arrest and enhances the cytotoxicity of cisplatin. We show here that UCN-01 at non-cytotoxic concentrations abrogated S and G2 arrest induced by cisplatin in two p53-defective human breast cancer cell lines. UCN-01 pushed the cells through S phase and mitosis, with subsequent apoptosis. Inhibition of mitosis with nocodazole reduced the apoptosis induced by UCN-01 plus cisplatin. Seven staurosporine analogs were compared for their ability to abrogate cell cycle arrest. Staurosporine was as effective as UCN-01 at abrogating S and G2 arrest, but the concentrations required were cytotoxic. K252a abrogated S phase arrest but failed to abrogate G2 arrest because alone it induced G2 arrest. Hence, K252a did not enhance cisplatin-induced cytotoxicity because it failed to push the cells through a lethal mitosis. None of the other analogs influenced cell cycle progression at the concentrations tested. Accordingly, UCN-01 was the only analog that overcame cell cycle arrest and enhanced the cytotoxicity of cisplatin while exhibiting no cytotoxicity of its own. Hence, UCN-01 remains the most promising candidate for testing clinically in combination with cisplatin.