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Objective To study the relationship between glutamine metabolism and radiotherapy resistance in nasopharyngeal carcinoma (NPC).Methods The glutaminase 1 (GLS1) knockout CNE1,6-10B cell lines and the control cell lines were established by lentivirus transfection technology.The CNE2 cell lines with GLS1 overexpression and control cell lines were established by liposome transfection technology.BPTES,one of Glutamine enzyme inhibitors,was used to inhibit the activity of GLS1.Glutamine/Glutamate(Gln/Glu) and Glu kits were used to detect glutamine metabolism in NPC cells;in vitro radiation clone formation experiment and flow cytometre mediated apoptosis assay were adopted to detect the effects of glutamine metabolism modulation on NPC radiosensitivity.Western blot and autophagic puncta monitor assay were carried out to detect the effects of glutamine metabolism modulation on autophagy status in NPC cells.Results Inhibiting the glutamine metabolism led to decreased clone formation,increased apoptotic rates,and decreased autophagic level in NPC cells;while activating the glutamine metabolism led to increased clone formation,decreased apoptotic rates and increased autophagic level in NPC cells.Conclusions The reduction of glutamine metabolism inhibits autophagy and increases radio sensitivity of NPC.
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Objective To investigate pathogenic factors,prevention approaches and therapeutic methods of neurosurgical postoperative intracranial infection.Methods A total of 89 cases were selected as our subjects from 1432 cases ho had received neurosurgical operation in the First People's Hospital of Shangqiu from April 2009 to April 2012.The clinical data of 89 cases with intracranial infection were retrospectively analyzed.Chisquare test was selected to analyze the factors which might cause infection.Effect of decision criteria was chosen to evaluate the cure effect.Results The infection rate was related to the approach to the post fossa,operation periods above 4 h,ventricular drainage,long indwelling drainage and cerebrospinal fluid (CSF) leakage (P ＜0.05 or P ＜0.01).However,the infection rate was not related to age,sex and application of antibiotics before the operation (P ＞0.05).As to effects,73 cases (82.02％) were cured,and 7 cases (7.87％) were showed sort of effects,as well as 8 cases(8.99％) were improved a little and 1 case(1.12％) with invalid.Conclusion It is important to adopt appropriate operation method,by decreasing operation time,preventing drainage from pollution carefully in order to prevent and decrease the intracranial infection after craniotomy,prevention CSF leakage and proper treatment could effectively cure intracranial infection.
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Objective To investigate the clinical effects of early cranioplasty and ventriculoperitoneal shunt in the treatment of traumatic brain injury.Methods 70 patients with traumatic brain injury were randomly divided into two groups.35 cases in the control group were treated with ventriculoperitoneal shunt,and received cranioplasty postoperative 3-6 months.35 patients in study group received early cranioplasty and ventriculoperitoneal shunt in 2-3 months after treatment.The therapeutic effects of the two groups were observed.Results After GCS,the excellent and good rate of study group was 71.4％,which was significantly higher than 57.2％ of the control group (x2 =7.47,P ＜ 0.05).The good rate of the study group was significantly higher than that of the control group (x2 =8.35,P ＜0.05).The moderate disability rate of the study group was significantly lower than that of the control group (x2 =7.33,P ＜0.05).The incidence rate of complications in the study group was 14.3％,which was significantly lower than 34.3％ in the control group (x2 =7.35,P ＜ 0.05).Conclusion Early cranioplasty and ventriculoperitoneal shunt therapy in the treatment of patients with traumatic brain injury can effectively improve clinical recovery of the patients,and reduce the postoperative complications.
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Objective To investigate the influence of early hyperbaric oxygen (HBO) therapy on cerebral edema and neural function in patients after minimally invasive surgery for intracerebral hemorrhage (ICH).Methods A random number table was used to divide 148 ICH surgery patients into a control group (n =75) and a treatment group (n =73).In the treatment group,HBO was administered in 51 cases 6-24 hours after surgery and then once a day for twenty days.Cerebral edema volume was measured by brain CT before the operation and on the 3rd,7th,14th and 21st day after the surgery.Neurological impairment was scored at the same time points.Results Average cerebral edema volume was significantly smaller in the treatment group than in the control group on the 7th,14th and 21st days,but not on the 3rd day.The neurological impairment scores (NIS) after therapy were significantly lower than that before therapy in both groups.The two groups' average scores were not significantly different before the operation or on the 3rd day,but they were significantly lower in the treatment group thereafter.Conclusion Early HBO therapy can significantly reduce cerebral edema and contribute to nerve functional recovery in patients after minimally invasive ICH surgery.
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OBJECTIVE@#To explore whether oncogenes DJ-1 and HSP27 are associated with invasiveness of human pituitary adenoma.@*METHODS@#Total proteins were extracted from samples of 20 invasive and 20 non-invasive pituitary adenomas and the expression of DJ-1 and HSP27 was analyzed by Western blot. The correlation of DJ-1and HSP27 with the invasiveness of pituitary adenoma was analyzed.@*RESULTS@#The strong positive rates of DJ-1 and HSP27 in the 20 invasive pituitary adenoma were 70% (14/20) and 80% (16/20), respectively. The invasive group had significantly higher expression of DJ-1 and HSP27 proteins than the noninvasive group [10% (2/20), 10% (2/20), respectively]. There was a positive correlation between the expression of DJ-1, HSP27 proteins and the invasiveness of pituitary adenoma as judged by the Spearman rank correlation test (P<0.05).@*CONCLUSION@#The proliferative activity and abnormal expression of oncogenes DJ-1 and HSP27 may play a significant role in tumorigenesis and progression of pituitary adenoma. There was a significant correlation between the expression of DJ-1 and HSP27 and the invasiveness of pituitary adenoma.
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Adult , Female , Humans , Male , Middle Aged , Young Adult , Adenoma , Metabolism , Pathology , Biomarkers, Tumor , Metabolism , Gene Expression Regulation, Neoplastic , HSP27 Heat-Shock Proteins , Genetics , Metabolism , Intracellular Signaling Peptides and Proteins , Genetics , Metabolism , Neoplasm Invasiveness , Oncogene Proteins , Genetics , Metabolism , Pituitary Neoplasms , Metabolism , Pathology , Protein Deglycase DJ-1 , Signal TransductionABSTRACT
Objective To construct an eukaryotic expression vector pEGFP-N1-CKB and establish a stably transfected NCI-H520 cell line.Methods Human CKB gene was amplified by PCR with human CKB cDNA library as the template and the fragment was combined with plasmid pEGFP-N1.The recombinant expression vector,pEGFP-N1-CKB,was transfected to NCI-H520 using Lipofectamin.The stably transfected cell line was established after G418 selection and the expression level of CKB gene before and after transfection was detected by Western blot.Results After identification by restriction enzyme digestion and sequencing,the eukaryotic expression vector,pEGFP-N1-CKB,was successfully constructed.The expression level of CKB in NCI-H520 transfected by pEGFP-N1-CKB was significantly higher than that in control.CKB gene had a stable transfection in NCI-H520 cells.Conclusions An eukaryotic plasmid encoding CKB (pEGFP-N1-CKB) has been constructed and a cell line expressed CKB stably has been successfully prepared.
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Objective To provide molecular genetic basis for oncobiological difference in left sided colon cancer and right sided colon cancer. Differentially expressed proteins in left sided colon cancer and right sided colon cancer were screened by proteomic technique. Methods Tissue samples including left sided colon cancer and right sided colon cancer were collected and preserved in the -80℃ refrigerator. In the first part of our experiment, protein was separated by 2-dimensional gel electrophoresis (2-DE) and the images of the gels were acquired by the scanner and then analyzed to find the differentially expression protein-spots in different groups. The peptide mass fingerprintings (PMF) was acquired by matrix assisted laser desorptiorn/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and the proteins were identified by data searching in the Mascotdatabase. Differentially expressed proteins were assayed by RT-PCR, Western blot, and immunohistochemical method. Results Altogether 55 differentially expressed protein spots were screened and 21 spots of them were identified. Compared with the right sided colon cancer, 14 proteins were up-regulated and 7 proteins down-regulated including HSP27 in the left sided colon cancer. HSP27 expressed higher in the right sided colon cancer than in the left sided colon cancer.Conclusion There are differentially expressed proteins in left sided colon cancer and right sided colon cancer, especially difference in HSP27 expression at mRNA and protein level, which may be molecular genetic basis for oncobiological difference in left sided colon cancer and right sided colon cancer.
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Objective To determine the significance and expression of S100A9 and NMP238 in cervical carcinoma with different concurrent chemoradiotherapy sensitivities. Methods Fresh carcinoma tissues were collected from untreated cervical carcinoma patients and preserved at -80 ℃. The tissues were classified into 2 groups:a high sensitivity group (HS) and a low sensitivity group(LS) according to their response to concurrent chemoradiotherapy. Protein was separated by 2-dimensional gel electrophoresis (2-DE). Peptide mass fingerprintings (PMF) were acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and the proteins were identified by data searching in the Mascot-database. Differential expressed proteins were assayed by Western blot and immunohistochemistry.Results Most of the gels were clear and were successfully and reproductively analyzed. Intensity and rate of S100A9 expression were higher in the HS group than in the LS group,and those of NMP238 expression were higher in the LS group than in the HS group. Conclusion S100A9 and NMP238 expression is associated with concurrent chemoradiotherapy sensitivity in cervical carcinoma.
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Objective To analyze the diagnostic value of 4 serum autoantibodies in nasopharyngeal carcinoma patients (anti-Rho-GDI-2, -HSP70, -CK19 and -LAP3 antibodies) using a serological proteomic approach. Methods The conditions for blocking, antigen concentrations and serum dilution were optimized to establish the ELISA method for detection of serum autoantibodies for Rho-GDI-2, HSP70, CK19 and LAP3. The serum concentrations of these autoantibodies were detected by the established method in 36 NPC patients, 20 other cancer patients and 20 healthy individuals. The statistical analysis was used to evaluate the diagnostic value of the 4 serum autoantibodies. Results The concentrations of 4 serum autoantibodies in the NPC patients were significantly higher than those in the healthy individuals(A450 values in NPC group of CK19, Rho-GDI-2, HSP70, LAP3 were 0. 188 8 ±0.047 3, 0.240 5 ± 0.024 7, 0. 116 8 ±0.025 3, 0.276 9 ±0.044 2 repectively. A450 values in healthy control group were 0.010 5 ±0.004 4, 0. 105 3 ±0. 016 9, 0. 098 6 ± 0. 014 0, 0. 149 8 ± 0. 033 1 respectively. F values were 4. 869, 15. 919, 10. 331,6. 369, respectively. P values were 0. 010, 0. 000, 0. 000, 0. 003, respectively). The sensitivities of antiRho-GDI-2, anti-HSP70, anti -CK19 and anti -LAP3 autoantibodies for diagnosis of NPC was 85.0%,75.0%, 75.0% and 75.0%, respectively, and the specificities were 58.9%, 80. 4%, 58. 9% and 57. 1%, respectively, anti-Rho-GDI-2 and anti-HSP70 achived the highest sensitivity and specificity respectively when single marker was analyzed. The sensitivity and specificity were 94. 4% and 95. 0%respectively when 4 markers was combined for analysis. Conclusion Detection of serum anti-Rbo-GDI-2,anti-HSP70, anti-CK19 and anti-IAP3 auotantibedios by ELISA may be of help in the screening and diagnosis for NPC.
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Objective To screen for new methylation association genes in HL-60 to reveal the pathogenesis of leukemia, and provide important theoretical and scientific basis for the prevention and cure of leukemia. Methods Two-dimensional fluorescence difference gel electrophoresis (F-2D-DIGE) was performed to separate the total proteins from acute myelogenous leukemia (AML) cell line HL-60 cells with or without 5-aza-2-deoxycytidine (5-aza-2-dC) treatment. Imaging software Decyder 6.5 and PDQuest were used to detect the differential expression protein spots, and matrix-assisted laser desorption/ionizaion time-of-flight mas spectrometer (MALDI-TOF MS) was adopted to identify the differential expression proteins. Results F-2D-DIGE maps of 5-aza-2-dC-untreated HL-60 and-treated HL-60 cells were established. A total of 53 differential protein spots were detected, and 35 differential proteins were successfully identified. Of the identified proteins, 32 proteins were up-regulated, and 3 proteins were down-regulated in HL-60 cells after 5-aza-2-dC treatment.Conclusion Thirty-five differential proteins may be associated with methylation in HL-60 cell line, which provides the important clues for epigenetic study of leukemia.
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The molecular techniques were used to analyse tyrosine phosphorylation of JAK2 and STAT3 in leptin receptor overpression cell lines and SH2-Bβ knockout (SH2-Bβ-/-) mice. The serum level of leptin in SH2-Bβ mice was measured by ELISA. The results showed that SH2-Bβ dramatically enhanced the leptin-stimulated tyrosine phosphorylation of JAK2 and STAT3 in vitro. Leptin-stimulated activation of JAK2 and phosphorylation of STAT3 were significantly impaired in hypothalamus of SH2-Bβ-/- mice. The fasting and postprandial serum levels of leptin and body weight were markedly increased in SH2-Bβ-/- mice. Therefore, SH2-Bβ is an endogenous enhancer of leptin sensitivity and regulates body weight via leptin/ JAK2/STAT3 pathway.
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OBJECTIVE@#To detect the methylation and expression of glioma pathogenesis-related protein 1(GLIPR1) gene in the acute myeloid leukemia (AML) cell lines and bone marrow cells from AML patients, and to determine the relationship between promoter methylation and expression of GLIPR1.@*METHODS@#Five leukemia cell lines, 54 bone marrows from the newly diagnosed AML patients, 48 bone marrows from the acute lymphoblastic leukemia (ALL )patients, 40 bone marrows from the chronic myeloid leukemia (CML) patients,35 bone marrows from control patients, and 8 bone marrows from the complete remission AML patients were collected. RT-PCR and methylation-PCR (MSP) were used to detect the mRNA expression and promoter methylation of GLIPR1, respectively, and the relationship between them was analyzed.@*RESULTS@#The level of GLIPR1 mRNA in the AML cell lines was lower than that in the chronic myeloid leukemia (CML) and ALL cell lines, whereas the methylation level of GLIPR1 in the former was higher than that in the latter. The level of GLIPR1 mRNA in the AML cell lines was significantly increased, but had no obvious changes in the CML and ALL cell lines after 5-aza-2dC treatment. The mRNA level of GLIPR1 in the AML bone marrows (0.38+/-0.20)was obviously lower than that in the ALL bone marrows (0.76+/-0.18), CML bone marrows (0.80+/-0.14), and control bone marrows(0.85+/-0.12). The level of GLIPR1 mRNA in the bone marrows with complete remission AML was obviously higher than that in the AML bone marrows before the treatment (0.78+/-0.13 vs. 0.36+/-0.20); but there was no obvious difference between the ALL bone marrows and the control bone marrows, and the CML bone marrows and the control bone marrows (both P>0.05). The positive rate of GLIPR1 gene methylation in the AML bone marrows (81.5%) was obviously higher than that in the ALL bone marrows(37.5%), CML bone marrows (27.5%) and the control bone marrows(14.3%). The positive rate of GLIPR1 gene in the bone marrows with complete remission AML was obviously lower than that in the bone marrows before the treatment (12.5% vs. 75.0%), but there was no obvious difference between the ALL bone marrows and between the control bone marrows,and the CML bone marrows and the control bone marrows (both P>0.05). There was a negative correlation between the mRNA level and methylation status of GLIPR1 in the AML bone marrows.@*CONCLUSION@#GLIPR1 expression is downregulated or even lost by promoter methylation in AML, and the expression and methylation level of GLIPR1 gene may have some significance in evaluating the curative effect of AML patients.
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Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Young Adult , DNA Methylation , HL-60 Cells , K562 Cells , Leukemia, Myeloid, Acute , Genetics , Metabolism , Neoplasm Proteins , Genetics , Metabolism , Nerve Tissue Proteins , Genetics , Metabolism , Promoter Regions, Genetic , RNA, Messenger , Genetics , MetabolismABSTRACT
OBJECTIVE@#To establish the protein expression map of nasopharyngeal carcinoma (NPC), and provide a basis for proteomic study of NPC.@*METHODS@#Laser capture microdissection (LCM) was used to isolate cancer cells from NPC tissues. Two-dimensional gel electrophoresis(2-DE) was used to separate the total proteins of LCM purified NPC cells. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was performed to identify the proteins, and bioinformatics was used to construct the 2-DE database of NPC proteome.@*RESULTS@#A 2-DE reference map of NPC was established. On the 2-DE map, a total of (1 312+/-30) protein spots were detected, and 427 protein spots representing 241 non-redundant proteins were identified. The 2-DE database of NPC proteome was constructed. These data could be accessed at our website (http://www.xyproteomics.org).@*CONCLUSION@#A protein expression profile of LCM purified NPC tissues has been established for the first time, which provides useful information and source for proteomic study of NPC.
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Humans , Computational Biology , Gene Expression Profiling , Lasers , Microdissection , Methods , Nasopharyngeal Neoplasms , Genetics , Metabolism , Neoplasm Proteins , Genetics , Metabolism , Proteome , Metabolism , Proteomics , Methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
OBJECTIVE@#To determine the effect of Ad-E1A gene therapy on in vivo radiosensitivity to nasopharyngeal carcinoma.@*METHODS@#CNE-2Z cells (2 x 10(5)) were subcutaneously injected into nude mice to develop tumor (1-3 mm) 6 days later. The tumor-bearing mice were then randomly divided into 6 groups (10 mice per group) for PBS treatment or treatment with radiotherapy, Ad-E1A, or Ad-beta-gal alone or radiotherapy in combination with Ad-E1A or Ad-beta-gal. The mice were treated with Ad-E1A or Ad-beta-gal (5 x 10(9) plaque forming units) by intratumoral injection twice weekly for 2 weeks at beginning of week 2. The mice treated with radiotherapy in combination with Ad-E1A or Ad-beta-gal received 2 Gy radiotherapy daily for 5 days following the first week of treatment with Ad-E1A or Ad-beta-gal. Control mice received PBS therapy or radiotherapy only after tumor cells were injected. When the size of tumor exceeded 2 cm, the mice were killed and the tumors underwent immunohistochemical analysis for VEGF and CD34 expression and TUNEL assay for apoptosis.@*RESULTS@#The growth delay time was longest in the Ad-E1A plus radiotherapy group. Tumors treated with Ad-E1A plus radiotherapy were 4.7-fold smaller than those treated with radiotherapy alone and 5.3-fold smaller than those treated with Ad-E1A alone. The survival rate of tumor-bearing mice treated with Ad-E1A plus radiotherapy was significantly higher than that of other treatment groups. The vessel density and the VEGF expression were significantly lower in tumors treated with Ad-E1A plus radiotherapy than those treated with radiotherapy alone, Ad-E1A alone, Ad-beta-gal alone, or Ad-beta-gal plus radiotherapy (P<0.01). TUNEL staining revealing apoptosis can be detected in the Ad-E1A group, radiotherapy group, Ad-E1A plus radiotherapy group, and more apoptosis can be detected in tumors treated with Ad-E1A plus radiotherapy than those of other treatment groups.@*CONCLUSION@#E1A gene therapy can effectively enhance the nasopharyngeal carcinoma sensitivity to the radiotherapy by down-regulating VEGF expression and inducing apoptosis.
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Animals , Humans , Mice , Adenovirus E1A Proteins , Genetics , Apoptosis , Physiology , Cell Line, Tumor , Genetic Therapy , Methods , Mice, Nude , Nasopharyngeal Neoplasms , Pathology , Radiotherapy , Neoplasm Transplantation , Radiation Tolerance , Genetics , Random Allocation , Vascular Endothelial Growth Factor A , Genetics , MetabolismABSTRACT
Objective To search for lymph node metastasis-associated proteins in human lung squamous carcinoma (hLSC).Methods Laser capture microdissection (LCM) was used to purify the target cells from lung primary tumor and matched lymph node metastatic tumor in hLSC. Two-dimensional gel electrophoresis (2-DE) was performed to separate the total proteins of microdissected tumor cells from lung primary tumor and matched lymph node metastatic tumor. PDQuest software was applied to analyze 2-DE images. Differential protein spots between the two types of tissues were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). The expression of Rho-GDIα, one of the differential proteins, in the microdissected lung primary tumor cells (LPTC) and matched lymph node metastatic tumor cells (LNMTC) was detected by Western blot. Results In the present study, 2-DE patterns of microdissected LPTC and LNMTC were established, and 22 differential proteins in the above two tissues were identified, of which 14 were down-regulated in LNMTC and 8 were up-regulated in LNMTC.Conclusion The 22 differential proteins may play some roles in the process of lymph node metastasis in hLSC, and the data provide new clues for metastasis-associated biomarker screen and mechanism of hLSC.
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Objective To identify proteins eliciting a humoral immune response in patients with nasopharyngeal carcinoma (NPC) by a serological proteome analysis, and provide candidate biomarkers for diagnosis and treatment of NPC.Methods Two-dimensional (2-DE) electropboresis was used to separate total cellular proteins from 19 NPC tissues.Separated proteins were transferred onto PVDF membranes and sera from 19 NPC patients and 19 healthy subjects were individually screened by western blotting for antibodies that react against separated proteins.Each tissue samples was subjected to three 2-DE gels and coomassie staining was performed in one of them.The protein spots which selectively reacted with the patient sera were excised from the preparative gels and subjected to further analysis of MOLDI-TOF MS and ESI-QTOF MS/MS.The proteins were identified based on peptide mass fingerprints and peptide sequence tags followed by searching database.Results In this study, 13 NPC associated antigens (HSP 70, HAS,HSP 60, CK 15, LAP 3, α-enolase, EBP 1, CK 19, ribosomal protein P 0, pyrovate dehydrogenase E1,guanine nucleotide-binding protein, prohibitin, Rho-GDI 2) that elicited an antibody response in most of NTC patients were identified.The positivities of these proteins were more than 21% all in NPC patients, but were lower or even absent in normal subjects.Conclusion These 13 NPC associated antigens and their autoantibodies may be useful for NPC diagnosis and treatment.
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Objective To establish high resolution, reproducible 2-dimensional electrophoresis (2-DE) profiles of invasive and non-invasive pituitary adenoma tissues and to identify differentially ex-pressed proteins between the invasive and non-invasive tissues. Methods The proteome from invasive and non-invasive pituitary adenomas tissues was dissected and analyzed by: (1) immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis, (2) silver staining, (3) imageMaster 2-D software analysis, (4) peptide mass fingerprint based (PMS) on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and (5) database comparison. Results High-resolution 2-D patterns of invasive and non-invasive pituitary adenoma tissues were successfully produced and re-peated 3 times for each sample. An average of 1080±24 and 1035±28 spots were detected for invasive and non-invasive pituitary adenoma tissues, respectively. Additionally, 975±45 and 918±56 spots were found to have an average matching rate of 90.3% and 88.7% for invasive and non-invasive tissues, re-spectively. The spot positional deviation was (1.563±0.259) mm for IEF and (1.088±0.206) mm for SDS-PAGE. A total of 99 spots of differential expression were matched between the invasive and non-in-vasive pituitary adenoma tissues. Thirty differential proteins, some of which were involved in the regula-tion of cells cycle and signal transduction, were initially characterized by PMS. Conclusion The acquisi-tion of well-resolved and reproducible 2-D patterns of invasive and non-invasive pituitary adenoma tissues and the identification of differentially expressed proteins provides a proteome database for invasive pituita-ry adenomas.
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Objective To investigate the methylation status of 14-3-3σ promoter in nasopharyngeal carcinoma cell lines and the influence of de-methylation treatment on 14-3-3σ expression. Methods Methylation status of 14-3-3σ gene promoter and 14-3-3σ mRNA expression were detected by methylation specific PCR (MSP) and RT-PCR in nasopharyngeal carcinoma cell lines CNE1, CNE2,5-8F,6-10B and immortalized nonneoplastic human nasopharyngeal epithelial cell line, NP69. Four nasopharyngeal carcinoma cell lines were treated with 5-asa-2' -deoxycytidine(5-aza-2dC) in different concentration for 72 h, then 14-3-3σ promoter meth-ylation status and m RNA expression were assessed, and western-blot was performed to detect the expression of 14-3-3σ protein. Results 14-3-3σ promoter methylation was detected by MSP in all of the four nasopharyn-geal carcinoma cell lines untreated by 5-aza-2dC whereas not in the treated ones or the immortalized human na-sopharyngeal epithelial cell line, NP69. Accordingly, 14-3-3σ mRNA expression was significantly discounted in untreated nasopharyngeal carcinoma cell lines as compared with NP69. 5-aza-2dC treatment dose-depend-ently reversed 14-3-3σ promoter methylation status and consequently upregulated the expression of 14-3-3σmRNA and protein in 4 nasopharyngeal carcinoma cell lines. High-differentiated CNE1 was more sensitive to 5-aza-2dC than lowly-differentiated CNE2, 5-8F and 6-10B. Conclusion Promoter methylation directly leads to decreased 14-3-3σ gene expression in nasopharyngeal carcinoma cell lines, and 14-3-3σ promoter de-methylation perhaps indicates a new target for nasopharyngeal carcinoma treatment.
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Objective To investigate the proteomics differences between the high-sensitivity(HS) and the low-sensitivity(LS)groups of cervical carcinoma treated by concurrent chemoradiotherapy,and to confirm the sensitivity associated proteins in intermediate stage and advanced cervical carcinoma.Methods Fresh carcinoma tissues were collected from 10 untreated cervical carcinoma patients.According to the response to concurrent chemoradiotherapy,the tissues were classified into HS group and LS group.In the first part of our experiment,protein separation was performed using two-dimensional gel electrophoresis(2-DE)with Amersham 18 am linear pH 3-10 immobilized pH gradient(IPG)strips.The images of the gels were analyzed by PD-quest 7.0 software to find the differentially expressed protein-spots in each group.Then the differentially expressed protein-spots were incised from the gels and digested by trypsin.The peptide mass flngerprintings(PMF)was acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).The proteins were identified by data searched in the Mascot-database.Two differentially expressed proteins were assayed by western blot and immunohistochemical methods.Results Most of the gels were clear and successfully analyzed by PD-quest 7.0 software.Most of the protein-spots concentrated on the area of 20-100 KDa(Mw)and pH4-8.The average number of the protein-spots was 781±74 in HS group and 766±52 in LS group.The match rate was 87.6%between the two groups.Eight proteins highly in HS group but lowly expressed in LS group included hemoglobin subunit beta,caspase-14 precursor,calmodulin-like,S100-A9 protein(MRP-14),galectin-7,HSKERC4,keratin 19 and actin.Ten proteins highly in LS group but lowly expression in HS group included anti HBs antibody light-chain Fab,laminB1,WARS protein,flavin reductase,glutamate dehydrogenase 1,nuclear matrix protein 238,retinal dehydrogenase 1,AFl 65172,subunit of replicative DNA polymerase and HSP70.The higher expression of HSP70 in LS group and galectin7 in HS groups were further confirmed by western blot and immunohistochemical method.Conclusions The 2-DE gels images are successfully acquired from high-sensitivity group and low-sensitivity group of intermediate stage and advanced cervical carcinoma tissues treated by concurrent chemoradiotherapy.Some differentially expressed proteins between the two groups can be further confirmed by western blot and immunohistochemical method.
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OBJECTIVE@#To determine the effect of Ad-E1A gene therapy on the radiosensitivity of nasopharyngeal carcinoma cell by downregulating the expression of VEGF in vitro.@*METHOD@#The human nasopharyngeal carcinoma CNE-2Z cell lines were investigated. The recombinant adenovirus vector containing E1A gene was used for this study. After CNE-2Z cells was treated with PBS, Ad-beta-gal and Ad-E1A for 48 h, the three groups were irradiated in different doses at 0, 2.4, 6, 8 and 10 Gy, the cytotoxicity was determined by MTT assay and cell cycle was analysis by flow cytometry. The VEGF expression were evaluated by RT-PCR assay and immunocytochemical analysis.@*RESULT@#Significant cell deaths by IR were observed in a dose dependent manner in the three group CNE-2Z cells. After transduction of the E1A gene into CNE-2Z cells, the sensitivity of these cells to radiation was enhanced than the PBS treated group and Ad-beta-gal treated group. Cell growth inhibition in Ad-E1A group by IR was strongly enhanced than Ad-beta-gal treated group and PBS treated group. RT-PCR assay and immunocytochemical analysis showed VEGF expression was downregulated in Ad-E1A treated group.@*CONCLUSION@#E1A gene therapy can effectively enhance the nasopharyngeal carcinoma cell sensitivity to the radiotherapy by down-regulating VEGF expression. These findings may pave the way for efficient radiation-gene therapy to NPC in future.