Identification of proteins involved in insulin stimulation in v ascular smooth muscle cells of SHR rats

AIM:To identify proteins involved in insul in stimulation and the molecular mechanism of proliferation and migration in vas cular smooth muscle cells (VSMCs).METHODS:A series of methods,including 2-D electrophoresis,PDQ uest software analysis of 2-DE gels,peptide mass fingerprinting based on matrix -assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TO F-MS) and SWISS-PROT database searching,were used to separate and identify the differentially expressed proteins.The difference of some proteins was proved by Western blotting.Proliferation and migration of VSMCs treated with insulin wer e also observed.RESULTS:DNA synthesis were increased in VSMCs.［3H］-thymidi ne incorporation in VSMCs from SHR (14.40±0.85) was higher than that in VSMCs from WKY (9.21±0.93,P<0.05).Migration rate of VSMCs from SHR was abou t 1.8 times higher than that in VSMCs from WKY (P<0.05).Image analysis re vealed that averages of protein spots detected were 502±32 and 612±39 in contr ol VSMCs and in cells treated with insulin,respectively.Result of western blo tting confirmed that α-SM protein was down-regulated and matrix Gla,OPN protei ns were up-regulated by insulin stimulation.CONCLUSION:The results suggest that the differential proteomic analysis may be useful to study related proteins involved in insulin-regulated p roliferation in VSMCs.     

Relationship between tumor metastasis suppressor gene KAI1 and the invasive and metastatic characteristics of osteosarcoma

AIM:To study the expression of metastasis suppressor gene KAI1 mRNA in osteosarcoma tissue and osteosarcoma cell lines,and the relationship between it and the biological behavior of the tumor cells.METHODS:RT-PCR was used to detect KAI1 mRNA in 18 cases of resected fresh osteosarcoma samples and three cultured osteosarcoma cell lines.The proliferative rate,the adhesive and invasive abilities of the 3 cell lines were detected.The results were treated by analysis system of images and analyzed with t test.RESULTS:The relative amount of KAI1 mRNA in osteosarcomas with lung metastasis was 0.80±0.50,while that was 1.48±0.64 in osteosarcomas without lung metastasis,the former was significantly lower than the latter (P<0.05).However,KAI1 mRNA had no corelation with the recurrence of osteosarcoma.The expression of KAI1 mRNA in U2-OS was highest (P<0.05),while the proliferation rate,the adhesive and invasive ability of U2-OS were the lowest among the 3 cell lines (P<0.01).CONCLUSION:The metastasis suppressor gene KAI1 might take part in influencing the lung metastasis of osteosarcoma,which might be caused by inhibiting the tumor cell proliferation,adhesion and invasion.     

Resveratrol enhances 5-fluorouracil-induced apoptosis of osteosarcoma CD133+ cell subset by promoting formation of Apaf-1/caspase-9 complex

AIM:To investigate the synergistic effect of resveratrol and 5-fluorouracil on osteosarcoma CD133⁺ cell subset.METHODS:Human osteosarcoma cell line MG-63 CD133⁺ cell subset and the corresponding CD133^- cell subset were treated with resveratrol and 5-fluorouracil.After treatment,the viability of MG-63 cells was measured by MTT assay.The apoptosis of MG-63 cells was analyzed by flow cytometry.Activation of caspase-9 and caspase-3,the expression of Apaf-1,and the release of cytochrome C were evaluated by Western blot.The interaction between Apaf-1 and pro-caspase-9 was detected by co-immunoprecipitation.RESULTS:The cell death and apoptosis of MG-63 CD133⁺ cell subset induced by 5-fluorouracil were significantly weaker than those in the corresponding MG-63 CD133^- cell subset.However,co-treatment with resveratrol significantly enhanced the effect of 5-fluorouracil on inhibiting the viability of MG-63 CD133⁺ cell subset.Mechanically,treatment with resveratrol upregulated the expression of Apaf-1.Transfection with Apaf-1 siRNA abolished the synergistic effect of resveratrol and 5-fluorouracil in MG-63 CD133⁺ cell subset.In addition,the results of co-immunoprecipitation indicated that the combination of resveratrol and 5-fluorouracil significantly induced the formation of Apaf-1/pro-caspase-9 complex,leading to the activation of caspase-9 in MG-63 CD133⁺ cell subset.CONCLUSION:Resveratrol enhances 5-fluorouracil-induced apoptosis of osteosarcoma CD133⁺ cell subset by promoting the formation of Apaf-1/caspase-9 complex.     

5-FU upregulates stem cell marker CD133 expression in colon cancer cells

AIM: To investigate the effect of 5-fluorouracil(5-FU)on the expression of the stem cell marker CD133 on colon cancer stem cells. METHODS: CD133 expression on several colon cancer cell lines was detected by flow cytometry. The CD133 positive cells from DLD1 cells were separated by the method of magnetic activated cell separation. Colony assay was used to measure self-renew ability and MTS assay was used to detect the sensitivity to 5-FU after separation. After 5-FU treatment, the change of CD133 mRNA level was measured by qPCR. RESULTS: CD133 expression on the surface of colon cacner cell lines DLD1, HT29, SW480, HCT116, Lovo, RKO was 30.20%, 82.00%, 0.34%, 91.80%, 85.30%, 0.28% respectively. DLD1 cells had two obvious populations according to CD133 expression. CD133 positive cells were separated from DLD1 cells, the positive purity was 87.21%±5.33% and the negative purity was 84.30%±4.65%. CD133 positive cells formed more colonies with limited dilution colony assay(46.33%±4.44% vs 31.00%±2.00%, P<0.05). CD133 positive cells were less sensitive to 5-FU compared to CD133 negative cells(20% less, P<0.01). 5-FU at concentration of 1 mg/L upregulated CD133 mRNA expression in both DLD1 and HT29 cells, the relative quantity was increased from 1 to 1.684±0.012(P<0.01)and 30.702±0.280 to 49.379±0.460(P<0.01)in HT29 and DLD1, respectively. CONCLUSION: Compared to CD133 negative cells, CD133 positive cells show more ability to form colonies in vitro, and are less sensitive to 5-FU. 5-FU upregulats the mRNA expression of CD133, resulting in the CD133 colon cancer stem cells enrichment during 5-FU treatment.     

Differential expression profiling of proteome for systemic lupus erythematosus with quantitative proteomic technique

AIM: To identify and quantify the total proteins in peripheral blood mononuclear cells (PBMC) of systemic lupus erythematosus (SLE) patients with quantitative proteomic technique, and to establish a differential expression profile of proteome for SLE. METHODS: Four-plex isobaric tags for relative and absolute quantification coupled with multiple chromatographic fractionation and tandem mass spectrometry were used to analyze the total proteins in PBMC from healthy controls and the patients of stable SLE, active SLE and rheumatoid arthritis. The proteins were identified by database searching with peptide mass fingerprinting. The differential expression of the proteins was compared. RESULTS: More than 400 proteins were identified. Compared with healthy controls, 44 proteins were discovered to be significantly expressed by more than 2 folds in stable SLE and active SLE, among which 9 proteins were up-regulated and 35 proteins were down-regulated. Compared with rheumatoid arthritis group, 52 proteins displayed 2 or more folds of changes in stable SLE and active SLE, including 19 up-regulated proteins and 33 down-regulated ones. The up-and down-regulated proteins between active SLE and stable SLE were 17 and 13, respectively. CONCLUSION: Quantitative proteomic technique is efficiently applicable for protein identification and relative quantitation in human peripheral blood mononuclear cells. Determination of the differentially expressed proteomic profile of SLE is helpful for better understanding the pathogenesis of SLE and developing new strategies for diagnosis and treatment of SLE.     

Ischemia reperfusion-induced proteomic changes in rat skeletal muscle

AIM: To investigate ischemia reperfusion (I/R)-induced proteomic changes in rat skeletal muscle. METHODS: Healthy male Wistar rats were randomly divided into two groups as follows (n=6): sham group and I/R group. I/R of right hind limb was induced by 4 h ischemia followed by 24 h reperfusion. The 2-DE was applied to separate the proteins extracted from skeletal muscle tissue at the end of experiment, followed by Coomassie Brillant blue R-250 staining. Computer image analysis was used to determine the differential expression of proteins between the two groups, and 7 protein spots expressed differentially were picked out and subjected to in-gel digest and MALDI-TOP for identification. RESULTS: 354±13 proteins were detected and the match rate was (78.7±1.4)%. 10 proteins displayed significant changes after I/R, of which, 6 proteins increased and 3 proteins decreased in expression. Moreover, 2 spots in I/R group were observed, only 1 spots of which in control. 5 proteins were identified after mass spectrometry. Mitochondrial aldehyde dehydrogenase (ALDH) precursor, heat shock 27 kD protein (HSP27), an unnamed protein product (increased in I/R group), α-actin (decreased in I/R group), and nuclear transport factor 2 (NTF-2) W7a mutant were found in I/R group. CONCLUSION: I/R injury induced differential proteomic changes in rat skeletal muscle. ALDH, α-actin and HSP27 expression, and NTF-2 mutation are involved in I/R injury.     

Isolation of endothelial progenitor cells from cord blood with CD133 immunomagnetic sorting

AIM: To isolate, purify and differentiate endothelial progenitor cells from cord blood in vitro and to study their biological characteristics. METHODS: CD133+ cells were selected from fresh cord blood mononuclear cells (MNC) by magnetic activated cell-sorting system (MACS). EPC was studied by flow cytometry, immunocytochemistry and immunofluorescence staining. Isolated cells were cultured in IMDM medium supplemented with or without VEGF, bFGF, SCF. RESULTS: The percentage of CD133+ cells of cord blood MNC was (1.41±1.14)%, and purity was 75%-85% (FACS method). CD133+ cells were grown on fibronectin-coated chamber slides in the presence of VEGF, bFGF, SCF. Within 1-2 hours of culture cells became adherent. On day 7-10, the adherent cells displayed a typical “cobblestone” morphology. After 14 days of culture, the adherent cells revealed a heterogeneous cell population, comprising small-sized round cells, spindle-like cells and formed tube-like structure. Weibel-Palade bodies were shown on the transmission electron microscopy photomicrographs. Compared with the original, cell markers CD133 and CD34 decreased significantly (77.0%±3.3% to 1.6%±2.2% and 93.1%±4.7% to 37.4%±4.9%, P<0.05), while Flk-1 increased significantly (from 22.3%±3.3% to 94.3%±4.1%, P<0.05) after 14 days of culture with VEGF, bFGF, SCF. The vWF was strongly expressed (77.9%±3.3%) on the 14th day later. CONCLUSION: Vascular endothelial progenitor cells were isolated from cord blood with specific expression of CD133/CD34/Flk-1. With the stimulation of the growth factors, seven-ten days after culture EPCs could be turned to endothelial cells.     

Antisense c-myc sensitizes osteosarcoma cells to cisplatin-induced apoptosis

AIM: To down-regulate expression of c-myc through antisense therapy and to investigate its effect on the sensitivity of osteosarcoma MG-63 cells to cisplatin-induced apoptosis. METHODS: The recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed and transfected into osteosarcoma MG-63 cells in vitro in order to down-regulate the expression of c-myc, and the change in the sensitivity to cisplatin-induced apoptosis was observed. MTT, Western blot, RT-PCR, flow cytometry (FCM) and electron microscope were used to evaluate tumor cell proliferation in vitro, genes expression related to apoptosis regulation and effects on the sensitivity of osteosarcoma MG-63 cells to cisplatin-induced apoptosis. RESULTS: Ad-Asc-myc down-regulated the expression of c-myc protein after transfected MG-63 cells for 48 h, combined with the treatment of 2.0 mg/L cisplatin for 2 h inhibited tumor cell proliferation in vitro by 38.0%. RT-PCR revealed that Ad-Asc-myc down-regulated the expression of Bcl-2 and up-regulated the expression of Bax. No appreciable change was observed in the expression of E2F-1. FCM showed that Ad-Asc-myc induced apoptosis in intransfected cells, and rendered it more sensitive to cisplatin. CONCLUSION: Antisense c-myc is able per se to induce apoptosis and sensitize osteosarcoma cells to cisplatin-induced apoptosis.     

The mitochondrial and structural protein changes in dexamethasone-induced mouse thymocyte apoptosis

AIM: To study mitochondrial mass and structural protein changes in dexamethasone (DEX)-mediated mouse thymocyte apoptosis process. METHODS: DEX-induced mouse thymocyte apoptosis model was established. Annexin V-FITC/PI double staining was used to identify apoptotic and necrotic cells by flowcytometry, JC-1 staining was adopted to test mitochondrial membrane potential (△Ψm), and cellular structural protein changes were studied with CFDA-SE staining. RESULTS: By 1×10-6 mol/L DEX stimulation, the apoptotic rate was 51.25%±5.51% and had significantly difference from control group (12.03%±2.00%); the necrotic rate in DEX group was 30.25%±3.67% and also had significantly difference from control group (10.11%±1.11%, P<0.01). Mitochondrial mass in DEX group ［FL1 (561.62±54.27)］ was significantly lower than that in control (900.25±38.80, P<0.01). DEX also caused significant down-regulation of △Ψm (P<0.01), FL2 in control group was 267.51±26.48, and in DEX group was 133.17±12.29. Mature T cells cultured for 48 h showed only one peak by CFDA-SE staining; while by Con A stimulation, there were three offspring peaks. Low FL1 cell cluster (5.25%±1.15％) exited in control group, while by DEX stimulation, this cluster significantly was larger (47.39%±9.76％, P<0.01). CONCLUSIONS: In mouse thymocytes, mitochondrial mass and cellular structural protein amount are reduced by DEX; CFDA-SE staining flowcytometry can served as an apoptosis detection method based on cellular structural protein amount change.     

Inhibitory effect of survivin antisense oligodeoxyribonucleotide combined with DDP in nude mice bearing human osteosarcoma xenograft

AIM:To investigate the feasibility and its mechanisms of improving therapeutic effect by antisense gene therapy combined with chemotherapy in osteosarcoma. METHODS:The human osteosarcoma implanted tumor model in the nude mice was established. By intratumoral injection and abdominal cavity administration, the tumor bearing mice were treated with survivin ASODN in combination with diamminedichloroplatinum (DDP) for a week. Comparison with each single-agent therapy and control group was performed in aspects such as tumor growth condition, pathological changes of tumor tissues;survivin protein expression in tumor tissues by immunohistochemistry, survivin mRNA expression levels by RT-PCR method and tumor apoptosis by Tdt-mediated dUTP nick end labeling (TUNEL). RESULTS:All nude mice survived the therapy. As compared with the control group, the antisense gene therapy group presented synchronous decrease in survivin mRNA and protein expression;all therapy group displayed tumor growth inhibition and cell apoptosis with different extent;while in contrast to single-agent therapy group, the combined therapy group showed stronger inhibition of tumor growth and abundant tumor cell apoptosis with the highest apoptotic rate. CONCLUSION:Synergistic effect was achieved by combination of DDP with ASODN that may overcome drug resistant of DDP and the combined strategy may shed new light on the cancer therapy.     

Enhancement effect of adenovirus mediated antisense c-myc gene and caffeine on chemotherapy of osteosarcoma cells to cisplatin

AIM: The cancer biology has showed that overexpression of oncogenes is responsible for the progression of human malignancies,antisense technology can block a certain gene expression.Caffeine has enhancement effect on chemotherapy of osteosarcoma cells to cisplatin,we constructed the recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment and investigated its effect on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin.METHODS: The recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed by cloning c-myc cDNA of about 750 base pairs in a reverse direction into adenovirus vector.Ad-Asc-myc and caffeine was used respectively or together to co-operate with cisplatin to treat the osteosarcoma MG-63 cells in vitro,and Western blotting,MTT,flow cytometry (FCM),electron microscope were used to evaluate expression of c-Myc protein,tumor cell proliferation in vitro,apoptosis and cell cycle analysis.RESULTS: Ad-Asc-myc was obtained with the titer of 2×10¹² pfu/L.Ad-Asc-myc down-regulated the expression of c-Myc protein,Ad-Asc-myc or caffeine enhanced the effects of 2.0,5.0 mg/L cisplatin on MG-63 cells.Moreover,Ad-Asc-myc combined with caffeine significantly enhanced this effects,not only on cisplatin-induced apoptosis,but also on tumor cells proliferation in vitro.The expression of bcl-2 was downregulated,bax were upregulated,while there was no change in the expression of E2F-1.FCM analysis showed that cisplatin treatment induced a block in S phase,and caffeine reversed this block and speeded up the progression of cells out of the S phase.Ad-Asc-myc induced obvious G₂/M phase arrest in transfected cells.CONCLUSION: Ad-Asc-myc combined with caffeine may enhance apoptosis-induced and chemotherapy effects of osteosarcoma MG-63 cells to cisplatin.     

Role of bcl-2 gene expression in inhabition of hepatocellular carcinoma by genistein in nude mice

AIM:To probe into the role of 1, 4, 5 - trisphosphate inositol (IP3) and bcl-2 gene expression in inhibiting hepatocellular carcinoma of nude mice by genistein. METHODS:Animals with hepatocellular carcinoma were treated with genistein 1 mg·kg-1·d-1 (ip) for 3 weeks. The volume and weight of tumaor were measured. IP3, bcl-2 mRNA, Bcl-2 protein were assayed by IP3-［3H］ Birtrak assay, RT-PCR, Western blotting, respectively. RESULTS:The tumor volume and weight of animals treated with genistein were lower than those in control (42.7mm3±27.8mm3 vs 52.3mm3±26.5mm3, 42.7mg±27.8 mg vs 91.3mg±31.4 mg). IP3 content was lower than that in control ［(13.4±1.4)nmol/g protein vs (35.3±6.6)nmol/g protein］. bcl-2 mRNA expression was lower in group treated with genistein than that in control (RI which was the gray degree multiply area of bcl-2 / the gray degree multiply area of β-actin 0.48±0.02 vs 0.56±0.15). Bcl-2 protein expression was lower in group treated with genistein than that in control (RI 1.69±0.52 vs 1.37±0.48). CONCLUSION:Genistein inhibits growth of transplanted hepatocellular carcinoma in nude mouse liver by reducing IP3 production and down-regulating bcl-2 gene expression.     

Analysis of expressive proteome in human hepatocarcinoma cell HepG2 transfected with miR-26a mimics

AIM: To determine whether microRNA-26a(miR-26a) is involved in development of liver cancer by analysis of proteomic expression profile of human hepatocarcinoma cell HepG2 transfected with miR-26a mimics.METHODS: HepG2 cells were cultured by a routine method and transfected with miR-26a mimics for 48 h for cell cycle analysis. The expressive proteome profiles of HepG2 cells with or without miR-26a mimics treatment were established by the methods of two-dimensional electrophoresis separation following lysis of the cells and extraction of the proteins. The proteomic expression profiles were analyzed by comparative proteomics technique to discover the important protein spots with differential expression. The identification of the proteins was conducted by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS). RESULTS: miR-26a brought down the proliferation of HepG2 cells. Total 11 protein spots with alteration of expressive amounts more than 2 times were successfully identified in the proteomic expression profile of HepG2 cells treated with miR-26a mimics, including annexin A1, peroxiredoxin 4, proliferating cell nuclear antigen, apolipoprotein A1, cytochrome C oxidase subunit 5A, cyclin E2, ribose-phosphate pyrophosphokinase 3, cyclin-dependent kinase 1 and phosphatidylethanolamine-binding protein 1. Among these, the expression of 3 protein spots was up-regulated and 8 of them was down-regulated.CONCLUSION: miR-26a contributes to the anti-cancer effect by expressive regulation of the proteins mentioned above, or directly or indirectly controls the proliferation, differentiation and death of hepatocarcinoma cells.     

Effect of miR-133 targeting NLRP3 on inflammatory activation of mouse Kupffer cells

AIMTo explore the effect of microRNA-133 (miR-133) targeting nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) on the inflammatory activation of Kupffer cells (KCs). METHODSThe KCs were isolated from mouse liver and identified. After successful identification, 1 mg/L lipopolysaccharide (LPS) was used to induce the KCs transfected with miR-133 inhibitor or miR-133 mimic. The mRNA expression levels of miR-133 and NLRP3 were detected by RT-qPCR. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in cell culture medium were measured by ELISA. The protein levels of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 were determined by Western blot. TargetScan was used to find the binding site of miR-133 and 3'UTR of NLRP3 mRNA, and the target relationship was identified by dual-luciferase reporter detection kit. RESULTSThe volume of KCs at 72 h was larger than that at 24 h, with clear boundary and stable shape. The result of carbon ink experiment showed that a large number of black particles were observed in the cells, which proved that the cells had strong phagocytic capacity and were KCs. After the KCs was induced by LPS at 1 mg/L, the level of miR-133 was decreased, while the expression of NLRP3 at mRNA and protein levels, the caspase-1 protein in the cells, and the levels of IL-1β and TNF-α in cell culture medium were increased (P<0.05). After transfection with miR-133 inhibitor, the level of miR-133 in the cells was decreased, while the expression of NLRP3 at mRNA and protein levels, the caspase-1 protein in the cells, and the levels of IL-1β and TNF-α in cell culture medium were increased (P<0.05). After transfection with miR-133 mimic, the level of miR-133 in the cells was increased, while the expression of NLRP3 at mRNA and protein levels, the caspase-1 protein in the cells, and the levels of IL-1β and TNF-α in cell culture medium were decreased (P<0.05). TargetScan analysis showed that the 3'UTR of NLRP3 mRNA contained the conservative bases of miR-133 sequence. Relative activity of luciferase in the cells transfected with miR-133 mimic was decreased (P<0.05). CONCLUSION miR-133 attenuates inflammation of mouse KCs by targeting NLRP3, thus protecting the KCs.     

CB2 receptor agonist JWH133 exerts protective effects on rat model of paraquat-induced acute lung injury

AIM: To study the protective effects of cannabinoid CB2 receptor agonist JWH133 on rat acute lung injury induced by paraquat (PQ).METHODS: Male Sprague-Dawley rats (n=72) were randomly divided into 4 groups. PQ group: PQ was administered intraperitoneally at the dose of 20 mg/kg; Low-dose JWH133 pretreatment group (L-JWH133 group): JWH133 (5 mg/kg, ip) was administered 1 h before PQ exposure; high-dose JWH133 pretreatment group (H-JWH133 group): JWH133 (20 mg/kg, ip) was administered 1 h before PQ exposure; control group: 1 mL saline was administered intraperitoneally. Arterial blood, bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected at 8 h, 1 d and 3 d after PQ exposure. PaO2 and the levels of TNF-α and IL-1β in BALF were measured via blood gas analyzer and ELISA, respectively. The pathological changes and lung injury scores were assessed at 3 d after PQ exposure. NF-κB and AP-1 protein levels were also determined by Western blotting.RESULTS: The decrease in PaO2, structural injury of the lung tissues, interstitial pulmonary edema, and the increase in IL-1β and TNF-α in BALF were observed in PQ-treated rats compared with control group. JWH133 pretreatment reduced the degree of lung tissue injury, decreased the levels of IL-1β and TNF-α in BALF and the NF-κB and AP-1 protein expression in the lung tissue compared with PQ group, especially in H-JWH133 group. CONCLUSION: CB2 receptor agonist JWH133 inhibits NF-κB and AP-1 protein expression in the lung tissues, and reduces the secretion of IL-1β and TNF-α in BALF after paraquat exposure, thus attenuating paraquat-induced acute lung injury.     

Diagnosis of colorectal cancer by WCX magnetic bead-based serum proteomic fingerprinting

AIM: To screen the potential protein biomarkers in serum for the diagnosis of colorectal cancer (CRC) by the technique of proteomic fingerprint.METHODS: Proteomic fingerprint combining magnetic beads with MALDI-TOF-MS was used to profile and compare the serum proteins from 98 patients with CRC and 80 healthy blood donors. Proteomic patterns associated with CRC were identified by Biomarker Wizard Software. The model of biomarkers was constructed and evaluated using the Biomarker Patterns Software.RESULTS: A total 68 discriminating m/z peaks were identified to be related with CRC (P<0.01). The model of biomarkers based on the 4 biomarkers (2 870.7, 3 084.0, 9 180.5 and 13 748.8) was constructed by Biomarker Patterns Software. Using this model, excellent separation between the CRC and the controls was generated. The sensitivity of the model was 92.85% (91/98) and the specificity was 91.25% (73/80). Blind test data indicated a sensitivity of 86.95% (40/46) and a specificity of 85.00% (34/40).CONCLUSION: The biomarkers for CRC can be discovered in serum by MALDI-TOF-MS combining the use of magnetic beads. The pattern of combined markers provides a powerful and reliable method for diagnosis of CRC with high sensitivity and specificity.     

Inhibition of hypothermic preservation-induced Smac/DIABLO protein expression by diazoxide in donor rat myocardium

AIM: To investigate the effect of a mitochondrial ATP-sensitive potassium channel (mitoKATP) opener diazoxide (DE) on Smac/DIABLO protein expressions in rat heart suffered from different duration of hypothermic preservation. METHODS: The Langendorff model of isolated rat heart was used. After stored in 4 ℃ Celsior solution with or without DE (30 μmol/L) for different time (0, 3, 6, 9 or 12 h). Cell apoptosis was detected by TUNEL technique. The expression of Smac/DIABLO protein in cytoplasm and total caspase-3 protein in myocardia tissue was also analyzed by Western blotting. RESULTS: (1) Compared to the hypothermic preservation groups, DE reduced the percentage of apoptotic cells and the expression of caspase-3 protein in myocardia tissue. (2) The peak of Smac/DIABLO protein expression level appeared at 6 h after hypothermic preservation, and which was postponed to 9 h by DE. (3) The above effects of DE were attenuated by a mitoKATP channel inhibitor 5-hydroxydecanoate (5-HD). CONCLUSION: The findings indicate that in the isolated rat heart, DE protects myocardium against different duration of hypothermic preservation injury via opening of mitoKATP channel and inhibition of Smac/DIABLO protein expression.     

Expression of PARP-1 in epithelial ovarian cancer and its relationship with epithelial-mesenchymal transition

AIM: To investigate the expression of poly(ADP-ribose) polymerase-1(PARP-1) in the epithelial ovarian cancer(EOC) and its relationship with epithelial-mesenchymal transition(EMT). METHODS: The expression of PARP-1, E-cadherin, vimentin and Snail was detected in the EOC and benign ovarian tumor tissues by immunohistochemical method and real-time PCR. The expression of PARP-1, E-cadherin, vimentin and Snail proteins in the SKOV3 cells treated with efficient PARP-1 inhibitor PJ34 was determined by Western blotting. RESULTS: The positive expression rates of PARP-1, vimentin and Snail were significantly higher in the EOC than that in the benign ovarian tumor tissues, whereas the positive expression rate of E-cadherin was the opposite(P<0.05). The expression of PARP-1, E-cadherin, vimentin and Snail in the EOC was associated with the histological grade, clinical stage and lymphatic metastasis(P<0.05), but no relationship with age and pathological types was observed. The expression of E-cadherin in the EOC was negatively co-related to that of PARP-1. In contrast, the expression of vimentin and Snail in the EOC was positively co-related to that of PARP-1. The relative mRNA expression of PARP-1, vimentin and Snail in the EOC was significantly higher than that in the benign ovarian tumor tissues(P<0.05), while the mRNA expression of E-cadherin in the EOC was remarkably lower than that in the benign ovarian tumor tissues(P<0.05). The protein expression of PARP-1, vimentin and Snail in the SKOV3 cells was significantly decreased(P<0.05), while E-cadherin protein was increased after treated with PJ34(P<0.05). CONCLUSION: PARP-1 may contribute to the onset of EMT in the EOC by regulating the expression of E-cadherin, vimentin and Snail. The role of PARP-1, which is relevant to EMT, might be important in the development of ovarian cancer.     

Identification of serum differential proteins in colorectal adenoma and early malignantly transformed adenoma by proteomics

AIM: To investigate the associated proteins and sensitive biomarkers for early diagnosis of colorectal adenocarcinoma by comparing the results of differential proteomic analysis between colorectal adenoma and early malignantly transformed adenoma. METHODS: Two-dimensional gel electrophoresis was used to define patterns of protein expressions of colorectal adenoma and early malignantly transformed adenoma. Proteins expressed differentially among groups were detected, cut out and analyzed by MALDI-TOF/TOF mass spectrometry. RESULTS: Two-dimensional protein maps of colorectal adenoma and early malignantly transformed adenoma were analyzed with gel-analysis software, an average of 1 672 spots in adenoma, 1 732 in early malignantly transformed adenoma were observed. 28 spots of a 1.5-fold change were found, including 15 proteins down-regulated and 13 up-regulated in early malignantly transformed adenoma, in which 23 proteins were identified by mass spectrometry, the rate of identification was 82.14%. 13 differential proteins were attained, 8 were up-regulated and 5 were down-regulated, which was classified to 6 categories, including protease inhibitor, complement, immunoglobulin, keratoproteins, signal transduction protein and function-unknown proteins. CONCLUSION: The changes of serum proteins in early malignantly transformed adenoma from adenoma can be identified by proteomic technology. Proteins detected in the study may provide new biomarkers correlated with biological behavior of colorectal adenocarcinoma.