OBJECTIVETo observe the cytotoxicity of indirubin derivative PHII-7 against human breast cancer MCF-7 cells and to study its primary mechanisms.

METHODSThe proliferation of MCF-7 cells was detected using MTT colorimetry. Annexin V/PI double staining was applied to detect the apoptosis rate of MCF-7 cells. The distribution of cell cycles was detected using PI staining and flow cytometry (FCM). The levels of reactive oxygen species (ROS) in MCF-7 cells were detected by DCFH-DA staining. The mRNA and protein levels of c-fos were detected using RT-PCR and Westem blot analysis.

RESULTSPHII-7 at different concentrations inhibited the proliferation of MCF-7 cells in a concentration-dependent manner, with the inhibitory rate ranging from 43.13% to 90.90% (P < 0.05). The inhibition was strengthened along with increased concentrations. PHII-7 at different concentrations could induce the apoptosis of MCF-7 cells. The early apoptosis rate was 1.43% +/- 0.02%, 9.14% +/- 0.36%, and 45.79% +/- 8.46%, respectively with the action of 1.25, 2.50, and 5.00 micromol/L PHII-7, respectively, showing dose-dependent manner. FCM analysis found that the proportion of MCF-7 cells in the G0/G1 phase and the S phase decreased after treatment with PHII-7, and the ratio of MCF-7 cells in the G2/M phase obviously increased (P < 0.01). The intra-cellular ROS level was significantly elevated 2 h after pretreatment with PHII-7. The levels of the protooncogene c-fos mRNA and protein were down-regulated in a dose-dependent manner after action of PHII-7.

CONCLUSIONSPHII-7 exerted obvious in vitro cytotoxic effects on MCF-7 cells. Its mechanisms might be associated with arresting the cell cycle, regulating the redox equilibrium, and down-regulating the expression of the protooncogene.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Female ; Humans ; Indoles ; pharmacology ; MCF-7 Cells

Animals ; Colon ; physiology ; Female ; Indoles ; pharmacology ; Male ; Myoelectric Complex, Migrating ; Rats ; Rats, Wistar ; Viscera ; physiology

OBJECTIVETo study whether necroptosis exists or not in neural cell death induced by aluminum.

METHODSSH-SY5Y cells were treated with 4 mmol/L AlCl(3) x 6H(2)O The cell viability was determined with CCK-8 kit after treated with Nec-1 at different dosages (0, 30, 60, 90 micromol/L). Mitochondria membrane potential (MMP), content of reactive oxygen species (ROS), and apoptotic rate/necrotic rates were measured with cytometry.

RESULTSNec-1 ameliorated the necrotic-like cell morphology, the cell viability were 0.28 +/- 0.05, 0.58 +/- 0.03, 0.68 +/- 0.04, and 1.03 +/- 0.17, there were significant differences between the Nec-1 treated groups and that of controls (t values were 3.25, 3.36, 4.56; P < 0.05). After Nec-1 treatment, the necrotic rates were 16.46% +/- 0.54%, 10.40% +/- 0.64%, 5.43% +/- 0.68%, and 6.28% +/- 0.35%, there were significant differences between the Nec-1 treated cells and that of controls (t values were 3.62, 7.32, 6.96; P < 0.05); while the apoptotic rates were 8.68 +/- 0.36, 7.66 +/- 0.53, 5.68 +/- 0.41, and 4.13 +/- 0.41, there was no significant difference among the groups (F = 6.33, P = 0.11). Cytometry had shown the increased cell MMPs after Nec-1 treatment, which were 67.54 +/- 6.36, 49.42 +/- 5.96, 84.79 +/- 6.86, and 95.51 +/- 7.01, there were significant differences as comparing MMPs of the middle and high dosage of Nec-1 treated cells with those of controls (t values were 3.21, 4.01; P < 0.05); while ROS contents in the Nec-1 treated SH-SY5Y cells were 54.07 +/- 3.32, 52.79 +/- 2.36, 54.68 +/- 1.91, and 59.23 +/- 2.96, there was no significant difference among the groups (F = 5.26, P = 0.19).

CONCLUSIONNec-1, as a specific inhibitor of necroptosis, might effectively block the cell death pathway induced by aluminum, it indicates that necroptosis should be one of the major causes of the SH-SY5Y cell toxicity induced by aluminum, and necroptosis also plays an important role in aluminum induced SH-SY5Y cell death.

Aluminum ; toxicity ; Apoptosis ; drug effects ; Cell Death ; drug effects ; Cell Line, Tumor ; Humans ; Imidazoles ; pharmacology ; Indoles ; pharmacology ; Neuroblastoma

OBJECTIVETo explore an efficient, stable system and method to verify the regulation effect of small molecule compounds on human hematopoietic stem cells (hHSC).

METHODSBy using combination of flow cytometry with study results of surface markers on hHSC, and optimation of sorting process for further studying the effect of small molecular compounds on stem property of hHSC, the single hHSC was treated with published small molecular compounds such as SR1 and UM171 which possess the expansion effect. After treating with hHSC for 14 d, the flow cytometric analysis of cell phenotypes and cell morphologic observation were performed, at the same time the hematopoietic function of cultured hHSC was verified by colony-forming cell (CFC) test and cobblestone area forming cell (CAFC) test.

RESULTSThe effects of SR1 and UM171 and their compositions in multi-cell culture were consistent with the published data, therefore the useful concentration of compounds were obtained. The results of multiparameter sorting of single cell (CD34+ CD38- CD45RA- CD90+ CD49f+) and ex vivo culture were consistent with the results of bulk cell culture. The results of cell phenotype analysis was in accordance with flow cytometric results. In addition, CFC test and CAFC test revealed that the colony-forming ability in treated group was significantly higher than that in control group (P<0.05).

CONCLUSIONThe rapid, efficient stably amplified and short-time culture system for single hHSC and method for varifying the effect of small molecular compounds are established, which provides platform for screening small molecular compounds and lays the foundation for further study of hHSC expansion.

Cell Culture Techniques ; Cell Separation ; Flow Cytometry ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Indoles ; pharmacology ; Pyrimidines ; pharmacology

The endophytic fungus Nigrospora sphaerica S5 derived from the semi-mangrove plant Myoporum bontioides was fermented. Its metabolites were purified by column chromatography. Nine compounds were obtained and identified as terezine P(1), 3-(1-hydroxyethyl)-4-methyl dihydrofuran-2(3H)-one(2), methylhydroheptelidate(3), hydroheptelidic acid(4), 5, 7-dimethoxy-4, 6-dimethylphthalide(5),(3R,4S)-(-)-4-hydroxymellein(6), pestalopyrone(7), indole-3-formaldehyde(8) and p-hydroxybenzaldehyde(9) by spectroscopic techniques. Terezine P(1) was a new alkaloid belonging to the terezine class with a pyrazine ring. Compounds 2-7 were lactones, of which 3 and 4 belonged to sesquiterpenes. Compounds 8 and 9 were indole alkaloids and phenols, respectively. Compounds 3-6 were purified from Nigrospora sp. for the first time. These compounds showed different degrees of antibacterial activity against Staphylococcus aureus, Escherichia coli of O6 serotype and E. coli of O78 serotype.
Alkaloids ; Anti-Bacterial Agents/pharmacology* ; Ascomycota/chemistry* ; Escherichia coli ; Formaldehyde ; Indoles/pharmacology* ; Lactones ; Molecular Structure ; Myoporum/microbiology* ; Phenols ; Pyrazines ; Sesquiterpenes

OBJECTIVETo investigate the effect of long-term administration of fluvastatin on improvement of ventricular remodeling of rats after myocardial infarction and its mechanism.

METHODSSprague-Dawley rats were subjected to ligation in anterior descending branch of coronary artery and treated with fluvastatin (20 mg.kg(-1) d(-1)) or distilled water for 8 weeks. Doppler echocardiography, hemodynamic study and cardiac histomorphometry were used to estimate the ventricular remodeling and cardiac function. Laser scanning confocal microscope was used to definite the distribution of superoxide anion (O(2)(*-)) and nitrogen monoxide. RT-PCR and immunohistochemistry were used to detect the expression of NOS2 and p22phox in mRNA and protein level. The level of lipid peroxidation, glutathione peroxidase, nitrogen monoxide and total cholesterol were detected too.

RESULTSAdministration of fluvastatin ameliorated left ventricular remodeling without affecting the infarct size [(40 +/- 6 vs 42 +/-5)%, P>0.05]. The level of left ventricular end-diastolic pressure [(18.24 +/-6.58 vs 10.74 +/-4.71) mmHg, P<0.05], right ventricular ameliorated relative weight [(0.92 +/-0.19 vs 0.71 +/-0.13) g/kg, P<0.05], the thickness of left ventricular posterior wall [(3.04 +/-0.28 vs 2.60 +/-0.36) mm, P<0.05] decreased after fluvastatin treatment. The left ventricular ejection fraction was not influenced, the relative lung weight and the left atrium diameter reduced [(5.79 +/-2.92 vs 3.69 +/-0.68) g/kg, (0.55 +/-0.12 vs 0.45 +/-0.04) mm, P<0.05]; the expressions of LPO in the plasma and myocardium [(8.64 +/-0.59 vs 7.71 +/-0.66) U/dl, P<0.05; (3.12 +/-0.38 vs 1.93 +/-0.40) ng/microg.pro, P<0.01] were reduced, and the overexpressed NO was inhibited [(436.87 +/-47.22 vs 313.78 +/-34.35) mg/dl, P<0.01], but the expression of GPx increased [(66.13 +/-8.31 vs 79.78 +/-2.38) mg/dl, P<0.01]. The expression of O(2)(*-) and the activity of NADPH oxidase subunit p22phox increased; NOS2 and its products NO were over-expressed too.

CONCLUSIONVentricular remodeling and hemodynamics are improved profoundly in MI rats treated with fluvastatin. The effect of antioxidative stress of fluvastatin might be involved in the mechanism.

Animals ; Antioxidants ; pharmacology ; Fatty Acids ; pharmacology ; Fatty Acids, Monounsaturated ; pharmacology ; Indoles ; pharmacology ; Male ; Myocardial Infarction ; metabolism ; pathology ; physiopathology ; Rats ; Ventricular Remodeling ; drug effects

The ocean continues to provide a plethora of unique scaffolds capable of remarkable biological applications. A large number of pyrroloiminoquinone alkaloids, including discorhabdins, epinardins, batzellines, makaluvamines, and veiutamine, have been isolated from various marine organisms. A class of pyrroloiminoquinone-related alkaloids, known as bispyrroloquinones, is the focus of this review article. This family of marine alkaloids, which contain an aryl substituted bispyrroloquinone ring system, includes three subclasses of alkaloids namely, wakayin, tsitsikammamines A-B, and zyzzyanones A-D. Both wakayin and the tsitsikammamines contain a tetracyclic fused bispyrroloiminoquinone ring system, while zyzzyanones contain a fused tricyclic bispyrroloquinone ring system. The unique chemical structures of these marine natural products and their diverse biological properties, including antifungal and antimicrobial activity, as well as the potent, albeit generally nonspecific and universal cytotoxicities, have attracted great interest of synthetic chemists over the past three decades. Tsitsikammamines, wakayin, and several of their analogs show inhibition of topoisomerases. One additional possible mechanism of anticancer activity of tsitsikammamines analogs that has been discovered recently is through the inhibition of indoleamine 2, 3-dioxygenase, an enzyme involved in tumoral immune resistance. This review discusses the isolation, synthesis, and evaluation of bioactivities of bispyrroloquinone alkaloids and their analogs.
Alkaloids ; chemistry ; pharmacology ; Animals ; Anti-Infective Agents ; chemistry ; pharmacology ; Antineoplastic Agents ; chemistry ; pharmacology ; Biological Products ; chemistry ; pharmacology ; Humans ; Indole Alkaloids ; chemistry ; pharmacology ; Indoles ; chemistry ; pharmacology ; Pyrroles ; chemistry ; pharmacology ; Quinolines ; chemistry ; pharmacology ; Quinones ; chemistry ; pharmacology

This study is to investigate the effect of fascaplysin on human cervical cancer cells (HeLa) in order to provide insights into the mechanisms of growth suppression involved in fascaplysin-mediated apoptosis. Cytotoxic activity of fascaplysin on HeLa cells was determined using MTT assay, cell cycle analysis, and apoptosis (Annexin V-FITC and PI double staining) studies. The role of the molecules in cell cycle regulation and apoptosis was analyzed by Western blotting and flow cytometry. Fascaplysin markedly inhibited HeLa cells proliferation in a dose-dependent manner, however, did not provoke G1 phase arrest in HeLa cells with downregulation of CDK4, cyclin D1 and CDK4-specific Ser795 pRb phosphorylation. Furthermore, fascaplysin induced significantly apoptosis evidenced by sub-G1 peak and Annexin V-FITC and PI double staining. The molecular mechanism of fascaplysin-induced apoptosis was characterized with the activation of caspase-3, -8, and -9, truncation of Bid, release of cytochrome c into cytosol, and down-regulation of Bcl-2 level. Fascaplysin exhibits anti-proliferation effect towards human cervical cancer HeLa cells through induction of apoptosis via extrinsic death pathway and mitochondrial pathway, but not arresting cell cycle progression at G1 phase. All together, these data sustain our contention that fascaplysin has anticancer properties and merits further investigation as a potential therapeutic agent.
Apoptosis ; drug effects ; Cell Cycle Checkpoints ; drug effects ; Cell Proliferation ; drug effects ; HeLa Cells ; Humans ; Indoles ; pharmacology ; Mitochondria ; metabolism

OBJECTIVETo investigate the effect of NSC348884, a nucleophosmin small molecular inhibitor, on the growth of hepatocellular carcinoma cell line HepG2 and its underlying mechanism.

METHODSAfter HepG2 cells were treated by NSC348884 for 4 days, the effect of HepG2 cells on proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay, the expression variation of nucleophosmin oligomer and monomer was measured using Western blotting, and cell apoptotic rate was detected by flow cytometry.

RESULTSThe proliferation of HepG2 cells was remarkably inhibited by NSC348884 treatment when the drug concentration ranged from 1 micromol/L to 10 micromol/L (P < 0.05), with a 50% inhibiting concentration of 1.4 micromol/L. After treatment for 24 hours, the expression level of nucleophosmin oligomer decreased obviously while that of nucleophosmin monomer increased (both P < 0.05). After treatment by 1 micromol/L and 2 micromol/L NSC348884, the 24-hour apoptotic rates of HepG2 cells were (13.770 +/- 0.335)% and (19.021 +/- 0.237)%, respectively, which were significantly higher than in the control group (6.950 +/- 0.207)% (P < 0. 05).

CONCLUSIONNSC348884 can promote the transformation of nucleophosmin oligomer to monomer and thus inhibit the growth of hepatic carcinoma cell line HepG2 in vitro.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Hep G2 Cells ; Humans ; Indoles ; pharmacology ; Nuclear Proteins ; antagonists & inhibitors ; metabolism

BACKGROUNDQacA, a main exporter mediating the multidrug-resistance of Staphylococcus aureus to a variety of antiseptics and disinfectants, possesses a topology of 14 alpha-helical transmembrane segments (TMS). Our study aimed to determine the importance and topology of amino acid residues in and flanking the cytoplasmic end of TMS5.

METHODSSite-directed mutagenesis was used to mutate 5 residues, including L146, A147, V148, W149 and S150, into cysteine. A minimum inhibitory concentration (MIC) and transport assay with or without N-ethylmaleimide (NEM) were performed to analyse the function of these mutants.

RESULTSAll of the mutants showed comparable protein expression levels. MIC analysis suggested that mutant W149C showed low resistance levels to the drugs, but the mutations at L146, A147, V148, and S150C had little or no effect on the resistance level. And the results of the fluorimetric transport assay were in agreement with those of MIC analysis, that is to say, W149C did not allow transport to the substrates to be tested, while the other mutants retained significant transport ability. The reaction of the different mutant proteins with Fluorescein-NEM revealed that the mutant L146C was highly reactive with NEM; the W149C and S150C mutants were moderately reactive; A147C was barely reactive and V148C showed no reactivity.

CONCLUSIONSThe study identified that residues W149 and S150 situated at the interface of the aqueous: lipid junction as functionally important residues, probably involved in the substrate binding and translocation of QacA.

Bacterial Proteins ; chemistry ; physiology ; Drug Resistance, Bacterial ; Ethylmaleimide ; pharmacology ; Indoles ; metabolism ; Membrane Transport Proteins ; chemistry ; physiology ; Structure-Activity Relationship