Effect of C/EBPalpha on the monocytic differentiation of HL60 cells induced by NSC67657 / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To figure out the function of C/EBPalpha in the monocytic differentiation of HL60 cells induced by a new steroidal drug NSC67657.</p><p><b>METHODS</b>The differentiation of HL60 cells was induced by NSC67657, and the cell surface antigen CD14 expression was detected by flow cytometry. The gene and protein expressions of CCAAT enhancer binding protein alpha (C/EBPalpha) before and after the induction of cell differentiation were determined by RT-PCR and Western blot. Eukaryotic expressing vector pDsRed-ICAT was constructed and transfected into HL60 cells, and its expression was verified. The effect of C/EBPalpha overexpression in HL60 cells was assessed by MTT assay, Wright's staining and flow cytometry before and after NSC67657 transfection.</p><p><b>RESULTS</b>HL60 cells could be induced into monocytes by 10 micromol/L ATRA within 5 days, and the coverage of CD14 positive cells reached 93.9% after 5 days of drug treatment. The eukaryotic expressing vector was successfully constructed, and over 90% positive clones were obtained after screening by G418 and electrotransfection. The results of proliferative analysis, chemical staining, ultrastructural observation, and CD11b detection confirmed that HL60 cells could be induced into granulocytic differentiation by overexpression of C/EBPalpha protein. Moreover, in the drug treatment group, transfected cells could not be induced into monocytic differentiation, and their granulocytic differentiation was also inhibited.</p><p><b>CONCLUSION</b>The monocytic differentiation of HL60 cells induced by NSC67657 may not be via the regulation by C/EBPalpha protein-mediated signal transduction. However, the overexpression of CEBPalpha may inhibit the process of NSC67657-induced monocytic differentiation in HL60 cells.</p>

Simultaneous determination of ibuprofen and pseudoephedrin hydrochloride and chlorpheniramine maleate in the compound buluoweimanamin tablets by HPLC / 南方医科大学学报

<p><b>OBJECTIVE</b>An HPLC method was developed to determinate Ibuprofen and Pseudoephedrine Hydrochloride and Chlorpheniramine Maleate in Compound BuluoWeimaNamin Tablets.</p><p><b>METHODS</b>Using HPLC with Kromasil C18 column, and acetonitrile -0.5% SDS- phosphate (580:420:1) as the mobile phase. The wavelength for detection was 262 nm.</p><p><b>RESULTS</b>Better linearities and good correlation coefficients were obtained: the concentration ranges of ibuprofen, pseudoephedrine hydrochloride and chlorpheniramine maleate were over 2.062-14.434 microg (r=0.9999), 0.296-2.072l microg (r=0.9999), and 0.0204~0.1428 microg (r=0.9998), respectively. The recoveries of ibuprofen, pseudoephedrine hydrochloride and chlorpheniramine maleate were 99.98% (RSD=0.52%), 99.72 (RSD=0.82%) and 99.545 (RSD=0.76%), respectively.</p><p><b>CONCLUSION</b>The method was convenient, accurate and specific. It can be used as a method to control quality of Compound Buluoweimanamin Tablets.</p>

A quantitative analysis of mitochondrial protein differential expressions in hydroxycamptothecin-treated hepatoma cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the differentially expressed mitochondrial proteins in hydroxycamptothecin (HCPT)-treated SMMC-7721 cells by using quantitative proteome.</p><p><b>METHODS</b>SMMC-7721 cell apoptosis was induced by HCPT and the mitochondria were isolated with a mitochondria isolation kit. Mitochondrial proteins labeled with a cleavable isotope-coded affinity tag were identified and quantified using two-dimensional liquid chromatography/tandem mass spectrometry.</p><p><b>RESULTS</b>Highly purified mitochondria were obtained. Seventy-four mitochondrial proteins, which were statistically significantly altered (P less than 0.05) in HCPT-treated cells, were identified and analyzed. A total of 42 proteins were significantly down-regulated, and 32 were up-regulated in the cells that responded to apoptosis. The functions of these proteins were likely involved in cell energy metabolism, nucleic acid translation and transcription, cytoskeleton, etc.</p><p><b>CONCLUSION</b>Our results about the information of differentially expressed mitochondrial proteins in HCPT-treated cells and the control cells will help to understand the mechanism by which HCPT induces cell apoptosis. The integrated techniques we used in this study will be helpful to the investigation of subcellular quantitative proteomics.</p>

Effect of total saponins of Rubus parviflolius (TSRP) on change of hydrated amount and blood-brain barrier in rats during focal cerebral ischemic/reperfusion / 中国中药杂志

<p><b>OBJECTIVE</b>To explore the effects of total saponins of Rubus parviflolius (TSRP) on brain edema and blood brain barrier in rats.</p><p><b>METHOD</b>The model of local cerebral ischemia was established in rats by reversible inserting a nylon thread into the anterior cerebral artery through the internal carotid artery brain hydrated amount and content change of Evan' s blue (EB) in cortex subjected to 2h middle cererbral artery occlusion (MACO) followed by 6 h, 24 h, 48 h, 72 h reperfusion and effect of TSRP. penetrability of blood brain-barrier (BBB) the index includes brain hydrated amount and penetrability of blood brain-barrier BBB.</p><p><b>RESULT</b>Com- pared with I/R group. Both brain hydrated amount and the EB content decreased significantly in TSRP groups on the 6 h, 24 h, 48 h, 72 h of reperfusion after 2 hour of cerebral ischemia induced by MACO model.</p><p><b>CONCLUSION</b>TSRP could decrease brain hydrated amount and markedly lower permeability of blood-brain barrier subjected to 2 h MACO followed by 24 h reperfusion, and this may be a mechanism of TSRP alleviating brain edema during I/R.</p>

Proteomic analysis of mitochondrial proteins in hydroxycamptothecin-treated SMMC-7721 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the differentially expressed mitochondrial proteins in hydroxycamptothecin (HCPT)-treated SMMC-7721 cells by comparative proteomic analysis.</p><p><b>METHODS</b>Apoptosis of SMMC-7721 cells were induced by using HCPT and their mitochondria were isolated with a mitochondria isolation kit for cultured cells. Three different solubility protein fractions were extracted with ReadyPrep Sequential Extraction Kit and were separated by two-dimensional gel electrophoresis (2-DE). PDQuest software was used to differentiate mitochondrial proteins between control cells and HCPT-treated cells. Matrix assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS) was used to identify some of the different proteins.</p><p><b>RESULTS</b>Highly purified mitochondria and high resolution 2-DE patterns of the proteins were obtained. Forty-four mitochondrial protein spots from the HCPT-treated cells showed different expressions compared to those of the control cells. Twenty of the different protein spots were analyzed by MALDI-TOF-MS.</p><p><b>CONCLUSION</b>Differently expressed mitochondrial proteins in HCPT-treated cells and control cells were obtained in this study. This will be of help to understand the mechanism by which HCPT induces cell apoptosis.</p>

Dynamic analysis of major active ingredients of Cortex Moutan cultivated in Dianjiang county of Chongqing / 中国中药杂志

<p><b>OBJECTIVE</b>To study the change of paeonol and paeoniflorin, the two major active ingredients contained in Cortex Moutan cultivated in Dianjiang county of Chongqing, due to the change of some influence factors, and explore suitable plant conditions and quality cotrol methods of Cortex Moutan.</p><p><b>METHOD</b>Paeonol and paeoniflorin were determined by HPLC in samples from Dianjiang.</p><p><b>RESULT AND CONCLUSION</b>The ratio of paeonol and paeoniflorin in Cortex Moutan was regularly influenced by altitude, the growth years and harvest time. Cortex could be cultivated at altitude of 400 m to 800 m but 600 m is the best aria because of the peak of the percentage composition of paeonol and paeoniflorin at 600 m. The first and middle third of October in the fifth year is the best picking time of Cortex Moutan because of the maximum of the percentage composition of paeonol and paeoniflorin.</p>

The preotective effects of total glycosides Rubus parviflolius on cerebral ischemic in rat / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the protective effects of total glycosides Rubus parviflolius (TGRP) on local cerebral ischemic.</p><p><b>METHOD</b>The local cerebral ischemia in rat was made by middle cerebral artery occlusion(MACO). The infraction weight was determined by TTC stain. SOD, MDA, GSH and apoptotis were determined with different method respectively.</p><p><b>RESULT</b>TGRP 20, 10 mg x kg(-1) ig markedly improved the abnormal nervous symptoms, incredsed the SOD, GSH activity and reduced contentes of MDA in brain of MACO rat, TGRP 20 mg x kg(-1) ig significantly decreased the numbers of apoptotic cells in ischemic cortex.</p><p><b>CONCLUSION</b>TGRP has protective effects against cerebral infraction, and its mechanism may be related to anti-apoptotis and free radical.</p>

Effect of hydroxycamptothecin on apoptosis-inducing factor (AIF) expression and on AIF translocation in human hepatocellular cancer cell SMMC-7721 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effect of hydroxycamptothecin (HCPT) on apoptosis-inducing factor (AIF) expression and AIF translocation from mitochondria to the nucleus in human hepatocellular cancer cell SMMC-7721 during apoptosis.</p><p><b>METHODS</b>After treatment with 80 mg/ml of HCPT, the cancer cells were stained with A0/EB to monitor their apoptosis. Their mitochondria was examined with electronmicroscopy and the AIF expression of the cells was tested by RT-PCR and Western blot. The translocation of AIF from mitochondria to the nucleus during apoptosis was analyzed by confocal microscopy.</p><p><b>RESULTS</b>SMMC-7721 cells treated with HCPT showed chromatin condensation, nuclear fragmentation and mitochondria swelling. The mRNA and protein expression of AIF in treated and untreated SMMC-7721 cells were not significantly different. However, cells treated with 80 mg/ml HCPT for 6 h or 12 h showed massive translocation of AIF into the nuclei.</p><p><b>CONCLUSION</b>These results show the important role the mitochondrial pathway of apoptosis plays in HCPT-induced tumor cell death, at least in SMMC-7721 cells.</p>

Two-dimensional gel electrophoresis of subcellular fractions of hepatoma cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To seek a better profiling of proteins of hepatoma cells.</p><p><b>METHODS</b>The homogenate of hepatoma cells QGY-7703 was fractionated into four parts by differential centrifugation: the nuclei, the pellet by 20,000 x g, the pellet by 100,000 x g and the cytosolic supernatant. The four fractions were submitted to two-dimensional gel electrophoresis and their electrophoretic patterns were analyzed.</p><p><b>RESULTS</b>In comparison with the protein pattern of hepatoma cells not fractionated, the patterns of the four fractions display many more protein spots, and a large number of proteins present in the nuclei and cytosolic supernatant were not shown in the not-fractionated samples.</p><p><b>CONCLUSION</b>Preparation of subcellular fractions before electrophoretic procedures proves to be very useful; not only can it improve the results of two-dimensional gel electrophoresis, but also can lead to research into the subcellular level.</p>

Study on differentially expressed molecules influencing the metastatic potential between highly and poorly metastatic human lung giant cell carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the metastasis-associated molecules differentially expressed in highly and poorly metastatic sublines and the mechanism of metastasis in lung giant cell carcinoma.</p><p><b>METHODS</b>Highly and poorly metastatic sublines (PLA801D and PLA801C)were used as metastasis model. Cell motility and invasion assay in vitro were first compared between the two sublines. Then, gelatin zymography analysis was used to determine the MMP-2 and MMP-9 activity. The protein expression level of secreted MMP-2, MMP-9, TIMP-1, TIMP-2 and intracellular expression level of p53, p16, PCNA, CD44(V6) isomeride, E-cadherin, CK18, nm23-H1 as well as the mRNA expression level of MMP-2, MMP-9, TIMP-1, TIMP-2, VEGF were compared through Western blot. Semi-quantitative RT-PCR analysis was used to determine the intracellular mRNA expression of MMP-2, MMP-9, TIMP-1, TIMP-2 and VEGF.</p><p><b>RESULTS</b>The in vitro cell invasion potential of highly metastatic subline PLA801D was significantly higher than that of poorly metastatic subline PLA801C by about 4 folds, while the cell motility potential was similar. The secreted MMP-2 activity was notably higher in PLA801D, which was initiated by the higher expression of MMP-2 at protein and mRNA level. In addition, the expression level of p53, PCNA, CK18 protein and VEGF mRNA were significantly higher, while the expression level of p16, E-cadherin and nm23-H1 protein were significantly lower in PLA801D. Some molecules such as MMP-9, TIMP-1, TIMP-2, CD44(V6) isomeride, which had been reported to be associated with tumor metastasis, were not observed to change significantly between the two sublines.</p><p><b>CONCLUSION</b>There are significant differences in metastatic potential and phenotypes between highly and poorly metastatic sublines of lung giant cell carcinoma. Some differentially expressed molecules might be playing roles in promoting or inhibiting metastasis of lung giant cell carcinoma, which may be useful to elucidate the mechanism of metastasis.</p>

Function of IL-18 in promoting metastasis of lung cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the function of IL-18 in promoting metastasis of lung cancer.</p><p><b>METHODS</b>The differential expression of IL-18 protein or mRNA level between highly and poorly metastatic sublines of human lung giant cell carcinoma metastatic model was detected by Western blot, semi-quantitative RT-PCR and northern blot analysis. The poorly metastatic PLA801C subline or highly metastatic PLA801D subline was transfected with constructed IL-18 sense or IL-18 antisense expressed plasmid by lipofectamine stable transfection technique. The metastasis-related effect mediated by IL-18, the metastatic phenotype differences, cell motility and cell invasion potential in vitro determined by MICS system and the expression level of metastasis-associated biomarkers detected by Western blot analysis, were compared between IL-18 stably transfectants and mock control, i.e. between PLA801C/IL-18(S) and PLA801C/pcDNA3.1, or between PLA801D/IL-18(As) and PLA801D/pcDNA3.</p><p><b>RESULTS</b>IL-18 was only present in highly metastatic PLA801D subline at either protein or mRNA level, which implied that IL-18 might play a role in promoting metastasis of lung cancer. After IL-18 sense expressed plasmid was transfected into poorly metastatic PLA801C subline, IL-18 fused protein with myc tag detected by Western blot analysis using either IL-18 or myc tag monoclonal antibody. In addition, cell motility ability in vitro was significantly increased about 3 times and E-cadherin protein was significantly down-regulated at about 50% in PLA801C/IL-18(S) transfectants compared with mock control. While IL-18 expressed plasmid was transfected into highly metastatic PLA801D subline, IL-18 protein and mRNA were simultaneously decreased by 30%. In addition, cell invasion ability in vitro was significantly decreased at about 75% and E-cadherin protein was significantly up-regulated in PLA801D/IL-18(As) transfectants compared with mock control.</p><p><b>CONCLUSION</b>IL-18 might play a role in enhancing tumor metastasis of lung cancer by down-regulating E-cadherin protein expression.</p>