To study the regulation of androgen receptor (AR) expression in human prostate cancer LNCaP cells by triptolide (TP) and the possible mechanism, by using qRT-PCR and Western blot, the AR mRNA and protein levels in TP treated LNCaP cells were detected, and the AR protein level in TP and NF-κB inhibitor treated LNCaP cells was also detected; a series of pGL3-AR promoter reporter gene vectors were built using restriction-free cloning method, and the vectors were employed to investigate the effects of TP on the transcriptional activity of AR promoter in LNCaP cells; the upstream proteins which may play regulatory roles were detected using western blot assay. After treated LNCaP cells with TP for 48 h, AR mRNA and protein expressions decreased with increasing TP concentration. The expression of AR target gene PART1 and prostate specific antigen (PSA) was also downregulated by TP treatment; a series of pGL3-AR promoter reporter vectors were constructed and validated by sequencing and luciferase activity; the results of dual luciferase reporter assay showed that TP downregulated AR at the transcriptional level; PI3K/AKT/NF-κB pathway which is associated with AR promoter activity was drowregulated by TP. In conclusion, our results demonstrated that the transcriptional activity of AR in LNCAP cells was downregulated by TP, and PI3K/AKT/NF-κB pathway may be involved in the regulation mechanism.
Cell Line, Tumor ; Diterpenes ; pharmacology ; Down-Regulation ; Epoxy Compounds ; pharmacology ; Genetic Vectors ; Humans ; Male ; NF-kappa B ; antagonists & inhibitors ; Phenanthrenes ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Promoter Regions, Genetic ; Prostate-Specific Antigen ; metabolism ; Prostatic Neoplasms ; metabolism ; RNA, Messenger ; Receptors, Androgen ; metabolism ; Signal Transduction ; Transcriptional Activation

AIMTo establish the method of HPLC-fingerprint analysis for the quality control of Dalbergia odorifera and identify its main constituents by HPLC-MS.

METHODSThe 37 hatches of samples were analyzed on a Phenomenex Luna C18 column with a gradient of acetonitrile and 0.3% aqueous acetic acid at a flow rate of 1.0 mL x min(-1) and detected at 275 nm. Furthermore, the typical samples were detected by HPLC-DAD-MS under negative ion mode.

RESULTS37 batches of D. odorifera samples were classified into three types based on the results of similarity analysis. According to the comparison of the tR, MS data and UV maximum absorbance (gamma(max)) values with the standards, 10, 7 and 2 phenolic components were identified in three types of D. odorifera extracts, separately.

CONCLUSIONThe method is repeatable and reliable, and it is capable of effectively controlling the quality of D. odorifera.

Benzopyrans ; chemistry ; isolation & purification ; China ; Chromatography, High Pressure Liquid ; methods ; Chromones ; chemistry ; isolation & purification ; Dalbergia ; chemistry ; Flavanones ; isolation & purification ; Isoflavones ; Molecular Structure ; Plants, Medicinal ; chemistry ; Quality Control ; Reproducibility of Results ; Spectrometry, Mass, Electrospray Ionization

This paper is aimed to report the development of a method for the determination of the binding rate of plasma protein with salvianolic acid B. In vitro, equilibrium dialysis method was used to imitate the binding process between salvianolic acid B and plasma protein, in vivo, ultrafiltration method was used and the binding rate with HPLC was determined. Plasma samples were treated with methanol to precipitate the protein, and the buffer solution was directly determined after filtering. The calibration curve of the buffer solution was linear in the range of 0.5-20 microg mL(-1). The calibration curve of the plasma was linear in the range of 2-200 microg mL(-1). The extract recovery was 68.6%-81.9%. RSDs of intra- and inter-day precisions were all less than 8.5%. The binding rates of plasma protein with salvianolic acid B in vitro was 75.2% and in vivo was 92.1%. This paper shows the high binding power of salvianolic acid B to plasma protein with high sensitivity, good reproduction, simple management and fulfilling the requirement.
Animals ; Benzofurans ; blood ; isolation & purification ; metabolism ; Blood Proteins ; metabolism ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Male ; Protein Binding ; Rats ; Rats, Sprague-Dawley ; Reproducibility of Results ; Salvia miltiorrhiza ; chemistry ; Sensitivity and Specificity ; Ultrafiltration ; methods

OBJECTIVETo investigate the changes of drug sensitivity of spindle poison-induced polyploid tumor cells to chemotherapeutic agents and its possible mechanism.

METHODSNocodazole in a dose of 100 ng/ml was used to induce polyploidization in a breast cancer cell line MDA-MB-231 cells. The polyploid cells (T-MDA-MB-231) were sorted by flow cytometry. The morphological changes and proliferation of T-MDA-MB-231 cells were compared with that of MDA-MB-231 cells. The cell growth inhibition was assessed by MTT assay. The cells were treated with paclitaxel, docetaxel, vincristine, epirubicin, 5-Fu, VP16 and oxaliplatin, respectively. Those cells were labeled with annexin V-FITC/PI and analyzed by flow cytometry. Bcl-2 was knocked down in T-MDA-MB-231 cells using SiRNA and their growth inhibition was evaluated by MTT assay to evaluate the reversing effect of Bcl-2-silencing on drug resistance.

RESULTSThe polyploid T-MDA-MB-231 cells grew in vitro continuously and maintained constant DNA content. They had a larger cell size, and grew more slowly than MDA-MB-231 cells. The IC(50(s)) of T-MDA-MB-231 cells were significantly higher than that of the MDA-MB-231 cells: paclitaxel: (6.37 ± 0.07) vs. (2.05 ± 0.83) µmol/L; docetaxel: (32.98 ± 1.48) vs. (11.95 ± 0.98) µmol/L; vincristine: (35.28 ± 1.66) vs. (14.58 ± 0.94) µmol/L; oxaliplatin: (19.07 ± 0.45) vs. (9.75 ± 1.05) µmol/L; 5-Fu: (85.49 ± 3.21) vs. (31.35 ± 1.51) µmol/L; and epirubicin: (0.53 ± 0.06) vs. (0.15 ± 0.01) µmol/L, (all P < 0.05). The IC(50(s)) of VP16 in T-MDA-MB-231 cells was (2.85 ± 0.50)µmol/L, significantly lower than the (12.20 ± 1.55) µmol/L in MDA-MB-231 cells (P < 0.05), and that of T-MDA-MB-231 cells after Bcl-2-knocked down by siRNA was (19.59 ± 0.48) µmol/L, significantly higher than the (12.20 ± 1.55) µmol/L in the MDA-MB-231 cells (P < 0.05). The IC(50(s)) of docetaxel of T-MDA-MB-231 cells after Bcl-2-knocked down by siRNA was (21.52 ± 0.68) µmol/L, significantly decreased and lower than that before Bcl-2 silencing (32.98 ± 1.48) µmol/L.

CONCLUSIONSOur results indicate that polyploid tumor cells induced by spindle poison Nocodazole are more resistant to most of chemotherapeutic drugs. Downregulation of Bcl-2 increases the sensitivity of polyploid cells to docetaxel. The high expression of Bcl-2 may be one of the drug resistance mechanisms of polyploid tumor cells. The polyploid tumor cells are relatively sensitive to VP16, suggesting that VP16 might be an effective candidate drug for treatment of chemoresistant polyploid tumors.

Antineoplastic Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Down-Regulation ; Drug Resistance, Neoplasm ; Epirubicin ; pharmacology ; Etoposide ; pharmacology ; Female ; Fluorouracil ; pharmacology ; Gene Knockdown Techniques ; Humans ; Inhibitory Concentration 50 ; Nocodazole ; pharmacology ; Organoplatinum Compounds ; pharmacology ; Paclitaxel ; pharmacology ; Polyploidy ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Taxoids ; pharmacology ; Vincristine ; pharmacology

OBJECTIVEThe influence of processing methods on chemical constituents in Radix Paeoniae Alba was observed.

METHODA HPLC method was used for analyzing the changes of eight major constituents, namely gallic acid, paeoniflorin sulfonate, catechin, paeoniflorin sulfonate, albiflorin, paeoniflorin, benzoic acid, pentagalloylglucose and benzoylpaeoniflorin, with the three processing procedures of decorticating, boiling and fumigating by burning of sulphur. Analysis was performed using a Zorbax SB-C18 column (4.6 mm x 250 mm, 5 microm) with the mixture of acetonitrile (A) and 0.015% phosphoric acid solution as mobile phase in gradient mode. The detection wavelength was set at 230 nm and the column temperature was at 30 degrees C.

RESULTExcept for gallic acid and pentagalloylglucose, the other constituents decreased during procedure of decorticating and boiling. Fumigating by burning of sulphur would produce a new compound, paeoniflorin sulfonate, which was a byproduct from the reaction of paeoniflorin with SO2.

CONCLUSIONThe significant changes were produced in chemical constituents of Radix Paeoniae Alba during three processing procedures. Therefore, the processing of Radix Paeoniae Alba should be strictly controlled and standardized.

Benzoates ; analysis ; chemistry ; Bridged-Ring Compounds ; analysis ; chemistry ; Chromatography, High Pressure Liquid ; Gallic Acid ; analysis ; Glucosides ; analysis ; chemistry ; Hot Temperature ; Hydrolyzable Tannins ; analysis ; Molecular Structure ; Monoterpenes ; Paeonia ; chemistry ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Sulfur ; Technology, Pharmaceutical ; methods

Objective To study the chest image appearances of penicilliosis marneffei(PSM)in patients with acquired immune deficiency syndrome(AIDS).Methods Chest imaging features of PSM in 36 patients with AIDS were retrospectively analyzed.Results Radiographic features of infiltrative lesions and focal lung consolidation were found in 14 cases(38.89%),in which 2 cases were with single lung disease(5.56%)and 12 cases with bilateral lung involvement(33.33%).Eight cases had diffuse lesions (22.22%),10 cases had reticular image patterns(27.78%),9 cases had nodular patterns(25.00%), 7 cases had ground-glass shadows(19.44%),6 cases had diffuse miliary lesions(16.67%),4 cases had enlarged bilar and enlarged mediastinum lymph nodes(11.11%).Cystic lesions was found in 5 cases (13.89%).Four cases had pleural effusion(11.11%),and 2 cases had nodular bump(5.56%). Pericardial effusion and pneumothorax each appeared in 1 case(2.78%).By HRCT,infiltrative lesion and focal lung consolidation were found in 32 patients(88.89%),in which 4 cases were with single lung lesions (11.11%)and 28 cases were with bilateral lung lesions(77.78%).Thirteen cases had diffuse lesions (36.11%),10 cases had pulmonary interstitial hyperplasia(27.78%),9 cases had nodular patterns (25.00%),8 cases had ground-glass shadows(22.22%),9 cases had diffuse miliary lesions(25.00%), 21 cases had enlarged lymph nodes in the mediastinum(58.33%).Cystic lesions were found in 8 cases (22.22%).Thirteen cases had pleural effusion(36.11%),and 2 cases had nodular bump(5.56%). Pericardial effusion and pneumothorax each appeared in 1 case(2.78%).Conclusion The image appearances of PSM in patients with AIDS include infiltrative lesions or focal lung consolidation,ground- glass shadow,enlarged hilar and mediastinum lymph nodes,pleural effusion,interstitial involvement or reticular image pattern(pulmonary interstitial hyperplasia),diffuse miliary lesion,and cystic lesion.

BACKGROUNDPim-1 plays an important role in the apoptosis, proliferation, differentiation of cancer cells and progression of cancer. In this study we detected the expression of pim-1 mRNA in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer (PCa) and explored its diagnostic value for PCa.

METHODSThe prostate tissues were collected from 23 patients with PCa, 37 patients with BPH, and 3 healthy volunteers. Pim-1 mRNA expression levels in these samples were determined by the quantitative real-time PCR (QRT-PCR). The differences of expression were calculated based on a standard curve.

RESULTSThe ratio of pim-1 mRNA to beta-actin in the normal prostate, BPH, and PCa were 1.05 +/- 0.04, 2.57 +/- 0.74 and 4.45 +/-0.63, respectively. The differences among PCa, BPH and NT were significant (P < 0.05, respectively).

CONCLUSIONDetecting pim-1 mRNA expression by QRT-PCR provides a reliable metric for the diagnosis of PCa.

Aged ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Prostate ; metabolism ; Prostatic Hyperplasia ; metabolism ; Prostatic Neoplasms ; diagnosis ; metabolism ; Proto-Oncogene Proteins c-pim-1 ; genetics ; RNA, Messenger ; analysis ; Sensitivity and Specificity

OBJECTIVETo observe the effect of tanshinone IIA (TS IIA) pretreatment on the expression of the inflammatory factor IL-1β and RelA mRNA in rats with focal cerebral ischemia.

METHODSA total of 100 adult male SD rats were randomly divided into 6 groups, namely the model, ischemic preconditioning (IPC), TSIIA preconditioning, TSIIA treatment, sham-operated, and blank control groups. In the former 4 groups, rat models of focal cerebral ischemia were established with corresponding treatments. The expressions of IL-1β and RelA mRNA in each group were detected using RT-PCR.

RESULTSAll the groups showed expressions of IL-1β and RelA mRNA with the exception of the blank control group. Compared to the model group, TSIIA preconditioning group, TSIIA treatment group, and IPC group all had significantly reduced expression of IL-1β and RelA mRNA (P < 0.05). The expressions were lower in IPC group than in TSIIA preconditioning group and TSIIA treatment group(P < 0.05), and no significant difference was found in the expressions between the latter two groups.

CONCLUSIONThe protective effect of pretreatment with TS IIA against cerebral ischemia is related to the reduction of IL-1β and RelA mRNA expressions.

Animals ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; therapeutic use ; Antioxidants ; pharmacology ; therapeutic use ; Brain Ischemia ; drug therapy ; metabolism ; Diterpenes, Abietane ; pharmacology ; therapeutic use ; Infarction, Middle Cerebral Artery ; drug therapy ; metabolism ; Interleukin-1beta ; genetics ; metabolism ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Reperfusion Injury ; prevention & control ; Transcription Factor RelA ; genetics ; metabolism

BACKGROUNDFenvalerate (FEN) has been demonstrated to be a reproductive toxicant in humans and rodents. However, little is known about whether short-term exposure to low-dose FEN produces reproductive toxicity.

METHODSWe administered FEN (0.009 375, 0.1875, 3.750, or 45.00 mg×kg(-1)×d(-1) by gavage for 30 days) to male ICR mice and compared reproductive toxicity parameters between groups receiving different concentrations of FEN. Reproductive toxicity was evaluated by computer-assisted semen quality analysis (CASA), chlortetracycline (CTC) assay, and histopathology.

RESULTSThe sperm morphology and testis histology of FEN-exposed mice (all doses) were similar to that in controlling mice. Exposure to FEN at a concentration of 0.1875 mg×kg(-1)×d(-1) decreased sperm path straightness (STR) and linearity (LIN) (both P < 0.05), but had no significant impact on average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), lateral amplitude (ALH), beat cross frequency (BCF), or progressive motility (MOT). FEN reduced the rate of mouse sperm capacitation in a dose-dependent manner.

CONCLUSIONThe present results demonstrate that exposure to low-dose FEN for 30 days reduces semen quality and sperm capacitation in adult mice.

Animals ; Body Weight ; drug effects ; Humans ; Male ; Mice ; Mice, Inbred ICR ; Nitriles ; pharmacology ; Organ Size ; drug effects ; Pyrethrins ; pharmacology ; Semen ; drug effects ; Semen Analysis ; Sperm Motility ; drug effects ; Testis ; drug effects

BACKGROUNDEarly diagnosis and timely treatment are important for improving therapeutic efficiency of prostate cancer. DNA array is a new bio-technology for disease diagnosis. This study was conducted to diagnose prostate cancer with cDNA macroarray and analysis gene expression profiles of some selective genes in prostate cancer.

METHODSTotal RNA was isolated from patients with prostate cancer and from normal people, and poly (A) RNA was further purified. Then it was analyzed for differentially expressed genes in prostate cancer and normal prostate by cDNA macroarray system.

RESULTSThere were different expressions in the nine prostate-associated specific genes in prostate cancer as compared with normal prostate, in which, 7 were significantly upregulated and 2 were down-regulated.

CONCLUSIONAs a diagnostic approach at molecular level, the cDNA macroarray is an effectively diagnostic method for prostate cancer.

Gene Expression Profiling ; Genes, Tumor Suppressor ; Humans ; Male ; Oligonucleotide Array Sequence Analysis ; Prostate-Specific Antigen ; blood ; Prostatic Neoplasms ; diagnosis ; genetics