Synthesis of a novel phosphate analog of 20-hydroxylecdysone with potent hypoglycemic activity.

A novel, water-soluble 20-hydroxylecdysono-20,22-phosphoric acid 2 and its sodium salt 3 were designed and synthesized from 20-hydroxylecdysone 1 in six steps and with 67% overall yield. The synthesized phosphoric acid 2 exhibited hypoglycemic activity >40-fold more potent than that of 20-hydroxylecdysone 1 at concentrations between 2 × 10⁻7 and 2 × 10⁻8 mol/l in a glucose consumption test in HepG2 cells. At a concentration of 2 × 10⁻9 mol/l, phosphoric acid 2 was still active, causing a maximum increase in glucose consumption of more than 500%, while 20-hydroxylecdysone 1 was inactive.

[Simultaneous determination of ibuprofen and pseudoephedrin hydrochloride and chlorpheniramine maleate in the compound buluoweimanamin tablets by HPLC].

OBJECTIVE: An HPLC method was developed to determinate Ibuprofen and Pseudoephedrine Hydrochloride and Chlorpheniramine Maleate in Compound BuluoWeimaNamin Tablets. METHODS: 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. RESULTS: 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. CONCLUSION: The method was convenient, accurate and specific. It can be used as a method to control quality of Compound Buluoweimanamin Tablets.

[Effect of C/EBPalpha on the monocytic differentiation of HL60 cells induced by NSC67657].

OBJECTIVE: To figure out the function of C/EBPalpha in the monocytic differentiation of HL60 cells induced by a new steroidal drug NSC67657. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Comparative proteomics analysis on differentiation of human promyelocytic leukemia HL-60 cells into granulocyte and monocyte lineages.

BACKGROUND AND OBJECTIVE: Human promyelocytic leukemia cell line HL-60 has potential to differentiate into granulocytes and monocytes by different inducers, such as NSC67657 and all-trans retinoic acid (ATRA). However, the mechanism is not clear yet. This study was to induce differentiation of HL-60 cells using ATRA and NSC67657, and compare the protein expression patterns using two-dimensional electrophoresis (2-DE). METHODS: HL-60 cells were cultured with ATRA and NSC67657 respectively. Cell proliferation was analyzed by MTT assay. Cellular surface specific antigen CD11b/CD14 was detected using flow cytometry (FCM) to assess cell differentiation. The alterations of cell morphology were observed with cellular chemical staining under electron microscope. Total protein was separated by modified 2-DE. The differentially expressed proteins were identified using PD-Quest software and analyzed by MOLDI-TOF MS. ICAT protein with differential expression was verified by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. RESULTS: The granulocytic and monocytic differentiation of HL-60 cells was induced by ATRA and NSC67657, respectively. The positive rates of both CD11b and CD14 in HL-60 cells were over 90% after 5-day treatment (2 micromol/L ATRA or 10 micromol/L NSC67657); cell morphology also represented characteristics of differentiation. Proteomic analysis showed that 25 proteins were differentially expressed with the same pattern in both differentiation groups, ten were differentially expressed only in monocytic differentiation group and 15 only in granulocytic differentiation group. Among them, ICAT expression was upregulated during NSC67657-induced differentiation of HL-60 cells. CONCLUSION: A batch of differentially expressed proteins has be found by 2-DE in HL-60 cells with granulocytic and monocytic differentiation, which would contribute to the following functional research on related proteins.

[Quantitative analysis of nuclear proteome in apoptosis hepatoma cells induced by hydroxycamptothecin].

The experimental foundation for investigating the pharmacological action of hydroxycamptothecin at subcellular quantitative proteomic level can be obtained depending on the information of differentially expressed nuclear proteins in hydroxycamptothecin-treated cells and control cells. The apoptosis was induced by hydroxycamptothecin in hepatoma cells, the nucleus of cells were isolated and verified with western blot. Nuclear proteins labelled with cleavable isotope-coded affinity tag (c-ICAT) reagent were digested and purified. The expression ratio of the identical nuclear protein derived from apoptosis cell and control cell can be gained using shotgun proteomic method based on multiple dimensional liquid chromatography-linear ion trap/orbitrap mass spectrometer combined with c-ICAT strategy. A total of 42 nuclear proteins were significantly (P<0.05) altered in hydroxycamptothecin-treated cells, among them, 12 proteins showed significantly down-regulation, and 30 proteins showed up-regulation compared with control cells. The function of these proteins were likely involved in life processes of cells such as proliferation, apoptosis, differentiation, energy metabolism, nucleic acid synthesis and metabolism, structure of cell skeleton.

[A quantitative analysis of mitochondrial protein differential expressions in hydroxycamptothecin-treated hepatoma cells].

OBJECTIVES: To investigate the differentially expressed mitochondrial proteins in hydroxycamptothecin (HCPT)-treated SMMC-7721 cells by using quantitative proteome. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Dynamic analysis of major active ingredients of Cortex Moutan cultivated in Dianjiang county of Chongqing].

OBJECTIVE: 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. METHOD: Paeonol and paeoniflorin were determined by HPLC in samples from Dianjiang. RESULT AND CONCLUSION: 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.

Changes in the protein spectrum of mitochondria isolated from hydroxycamptothecin-treated hepatoma cells.

As one of the most potent topoisomerase inhibitors, hydroxycamptothecin is more active and less toxic than conventional camptothecin. Recently, we found that hydroxycamptothecin can induce cell apoptosis via the mitochondrial pathway. This study was designed to investigate the mitochondrial protein profile in HCPT-treated cells using high-accuracy and high-sensitivity protein-identification technology. Of the 39 mitochondrial protein spots investigated, 25 displayed elevated and 14 suppressed abundance in hydroxycamptothecin-treated cells. The 25 spots were identified by mass spectrometry and they included proteins involved in many essential cellular functions. The potential role of these proteins in hydroxycamptothecin-mediated apoptosis is also discussed. This study has produced a short list of mitochondrial proteins that might hold the key to the mechanism by which hydroxycamptothecin induces mitochondrial dysfunction and cell apoptosis. It has laid the foundation for further elucidating the role of hydroxycamptothecin during apoptosis. Successful applications of multiple techniques including two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Western blot analysis have demonstrated that proteomic analyses provide appropriate approaches for understanding of the roles of anticancer drugs.

[Cloning and expression of recombinant mitochondrial Delta1-120 apoptosis-inducing factor and its enhancive effect on apoptosis of hepatocellular carcinoma cell line SMMC-7721].

BACKGROUND & OBJECTIVE: Mitochondria play a key role in cell apoptosis, and apoptosis-inducing factor (AIF) is a kind of apoptotic protein located in mitochondria. The research on mitochondrial protein can be helpful for elucidating the role of mitochondria in apoptosis. This study was to clone and express recombinant human Delta1-120 AIF and validate its biological activities of binding DNA and inducing nuclear apoptosis. METHODS: A human AIF gene fragment of 1515 bp(mitochondrial localization sequence was deleted) was amplified from SMMC-7721 cells by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pET32a(+) vector to construct recombinant plasmid pET32a-AIF. The recombinant plasmid was transfected into E.coli BL2l (DE3). AIF expression was induced by isoprophylthio-beta-D-galactoside (IPTG), and detected by SDS-PAGE and Western blot. AIF protein was purified by Ni afinity chromatography and then renatured. The biological activity of renatured AIF protein was detected by electrophoretic mobility shift assay (EMSA) and Hoechst staining. RESULTS: The 1.5 kb AIF gene was successfully isolated, and cloned into pET32a(+) vector. Plasmid pAIF was identified by restrictive enzyme analysis and sequencing. Recombinant E.coli. strains expressing AIF were obtained. AIF protein amounted to 11% of the total bacterial protein when induced with IPTG at 37 degrees Celsius for 4 h. AIF was specially recognizing by anti-AIF and anti-his antibody. The purity of purified protein reached over 95%. After renaturation, AIF protein binded DNA and induced nuclear apoptosis. CONCLUSION: AIF protein with high purity and biological activity was obtained by the method described above.

[Proteomic analysis of mitochondrial proteins in hydroxycamptothecin-treated SMMC-7721 cells].

OBJECTIVE: To investigate the differentially expressed mitochondrial proteins in hydroxycamptothecin (HCPT)-treated SMMC-7721 cells by comparative proteomic analysis. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Effect of total saponins of Rubus parviflolius (TSRP) on change of hydrated amount and blood-brain barrier in rats during focal cerebral ischemic/reperfusion].

OBJECTIVE: To explore the effects of total saponins of Rubus parviflolius (TSRP) on brain edema and blood brain barrier in rats. METHOD: 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. RESULT: 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. CONCLUSION: 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.

[Proteomic approach to the effect of epirubicin on hepatoma cells at subcellular level].

Epirubicin is an antineoplastic agent known as an anthracycline. It acts directly on DNA by blocking its replication and transcription, so apoptosis can be induced for cancer cells. The protein expression of cancer cells will be altered due to the induction of pharmacological action of epirubicin, so it is important to pay attention to the altered profiling of proteins. Here proteomic strategy was applied to the hepatoma cells at subcellular level, comparative proteome analysis of mitochondria and nucleus were conducted between the hepatoma cells administered with epirubicin and the not-administered. Centrifugation was used for the subcellular fractionation, then 2-DE for the separation of proteins, imaging analysis for the diction of expression-altered spots, and MALDI-TOF-MS for the identification of proteins. In total, 15 proteins were found to have altered their expression after the induction of epirubicin, among them, 5 proteins showed up-regulated expression and 10 showed down-regulated expression. These altered proteins are involved in life processes of cells such as energy metabolism, protein biosynthesis, structure of cell skeleton, processing and maturation of mRNA, heat shock of cells and apoptosis.

[Comparative proteome analysis of hepatoma cells at subcellular level].

The subcellular proteome strategy can complement the separation power of two-dimensional electrophoresis, a step for subcellular fractionation is introduced before electrophoresis is conducted, and more proteins will be displayed in two-dimensional gels. A comparative analysis of proteomic profiling of mitochondria and nuclei was conducted between hepatoma cell and hepatocyte, in order to find more information involved in cancer development. The cultured hepatoma cell line QGY-7703 and hepatocyte line LO2 were used as research models in this work. Subcellular fractionation for mitochondrion and nucleus were done by ultracentrifugation, then two-dimensional electrophoresis was applied to the separation of mitochondrial and nuclear proteins, imaging analysis for the selection of differentially-expressed protein spots, and MALDI-TOF-MS for the identification of proteins. 54 spots from electrophoresis gels were selected as differentially-expressed protein for MS analysis which resulted in identification for 22 proteins. Among the 22 differentially-expressed proteins, 17 show a up-regulated expression and 5 show a down-regulated expression. These differentially-expressed proteins found in this work have a wide coverage of functions which are related to energy metabolism, cytosheleton, protein biosynthesis, pre-mRNA splicing and processing, apoptosis regulation. These results imply that cancer cell has experienced a fundamental change in structure and metabolism pattern.

Hydroxycamptothecin-induced apoptosis in hepatoma SMMC-7721 cells and the role of mitochondrial pathway.

The camptothecin (CPT) derivative hydroxycamptothecin (HCPT) containing 10-hydroxy represents one of the most potent topoisomerase I inhibitors described. This anticancer agent, currently undergoing clinical trials on gastric tumours, has been shown more active and less toxic than conventional camptothecins. To shed light on the mechanism of action of HCPT at the cellular level, we examined cell growth, apoptosis, changes of mitochondrial membrane potential, cytochrome c and AIF translocation in cancer cells by exposing these cells to HCPT for indicated time. The effect of HCPT on cell proliferation was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay and apoptosis was measured using flow cytometry, fluorescence microscopy and electron microscopy. Changes of mitochondrial membrane potential were monitored by fluorescence microscope. Western blot analysis was used to evaluate the release of mitochondrial cytochrome c and AIF; On the other hand, translocation of cytochrome c and AIF from mitochondria to cytosol during apoptosis were confirmed by confocal microscopy. HCPT could noticeably inhibit the proliferation of SMMC-7721cells and the IC(50) dose was about 0.22 microM; SMMC-7721 cells treated with HCPT showed typical characteristics of apoptosis rather than necrotic including phosphatidylserine (PS) exposed from the inner to the outer leaflet of the plasma membrane, abnormal cell morphology, chromatin condensation and nuclear fragmentation; On the other hand, during process of cell apoptosis, mitochondrial transmembrane potential was reduced; Compared with the control group, the mRNA and protein expression of cytochrome c and AIF in treated and untreated SMMC-7721 cells were not significantly changed (not shown). However, when cells were treated with HCPT, the massive translocation of cytochrome c and AIF to the nucleus was evident. Our results indicate that HCPT can inhibit proliferation and induce apoptosis of human hepatoma SMMC-7721 cells. Mitochondrial pathway of apoptosis, especially for cytochrome c and AIF translocation, may play an important role in apoptosis induced by HCPT.

[Effect of hydroxycamptothecin on apoptosis-inducing factor (AIF) expression and on AIF translocation in human hepatocellular cancer cell SMMC-7721].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: These results show the important role the mitochondrial pathway of apoptosis plays in HCPT-induced tumor cell death, at least in SMMC-7721 cells.

[The preotective effects of total glycosides Rubus parviflolius on cerebral ischemic in rat].

OBJECTIVE: To observe the protective effects of total glycosides Rubus parviflolius (TGRP) on local cerebral ischemic. METHOD: 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. RESULT: 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. CONCLUSION: TGRP has protective effects against cerebral infraction, and its mechanism may be related to anti-apoptotis and free radical.

[Two-dimensional gel electrophoresis of subcellular fractions of hepatoma cells].

OBJECTIVES: To seek a better profiling of proteins of hepatoma cells. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Differential expression of mitochondrial proteins in hepatoma cells analyzed using two-dimensional electrophoresis].

BACKGROUND & OBJECTIVE: Mitochondria play a key role in cell apoptosis. Proteomic analysis of mitochondria will contribute to the discovery of tumorigenic mechanism, early detection of cancer, and findings of anticarcinogens. This study was to identify the differentially expressed mitochondrial proteins in hepatoma cells using two-dimensional gel electrophoresis (2-DE). METHODS: Mitochondria were isolated from normal hepatocytes L02, and hepatoma QGY-7703 cells before and after treatment of epirubicin by density gradient centrifugation; the proteins of mitochondrial samples were analyzed with 2-DE. RESULTS: After density gradient centrifugation, the mitochondrial marker enzyme activities were increased by 11.8, 13.8, and 10.2 times, respectively, for L02 cells, untreated QGY-7703 cells, and epirubicin-treated QGY-7703 cells, and the 2-DE profiles identified 206, 217, and 214 protein spots, respectively, from the 3 samples. Compared L02 cells and QGY-7703 cells, 49 differentially expressed protein spots were identified; compared QGY-7703 cells before and after treatment of epirubicin, 29 differentially expressed protein spots were identified. CONCLUSION: A set of differentially expressed protein spots are detected in mitochondria of QGY-7703 cells with density gradient centrifugation and 2-DE.

[Study on differentially expressed molecules influencing the metastatic potential between highly and poorly metastatic human lung giant cell carcinoma].

OBJECTIVE: To study the metastasis-associated molecules differentially expressed in highly and poorly metastatic sublines and the mechanism of metastasis in lung giant cell carcinoma. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Function of IL-18 in promoting metastasis of lung cancer].

OBJECTIVE: To study the function of IL-18 in promoting metastasis of lung cancer. METHODS: 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. RESULTS: 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. CONCLUSION: IL-18 might play a role in enhancing tumor metastasis of lung cancer by down-regulating E-cadherin protein expression.