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protective effects of proanthocyanidins on beta-amyloid peptide (25-35)-induced pC12 cell apoptosis by blocking S-phase and increasing p53 gene expression

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Neural Regeneration Research 2010年第2期5卷 108-112页

作者： Hanfang Mei Zhaoyang Xie Qifeng Zhu Department of Biochemistry and Molecular Biology Guangdong Pharmaceutical University Guangzhou 510006 Guangdong Province China School of pubfic Health and Tropical Medicine Southem Medical University Guangzhou 510515 Guangdong Province China Institute of Biochemistry and Molecular Biology Guangdong Medical College Zhanjiang 524023 Guangdong Province China

BACKGROUND： Current studies related to the effects of proanthocyanidins on Alzheimer＇s disease have focused primarily on the signal transduction pathway of cellular apoptosis. However, the influence of p53 gene expression on cell cycle regulation, with regard to the protective mechanisms of proanthocyanidins, has not been reported. OBJECTIVE： To observe the effect of proanthocyanidins on cell cycle distribution, cellular apoptosis and p53 gene expression in β-amyloid peptide （25-35）（Aβ25-35）-induced pC12 cells cultured in serum-free media, and to investigate the molecular neuroprotective mechanisms of proanthocyanidins with regard to cell cycle regulation. DESIGN, TIME AND SETTING： A parallel, controlled, at the Institute of Biochemistry and Molecular Biology cellular, and molecular study was performed Guangdong Medical College from July 2006 to July 2008. MATERIALS： proanthocyanidins were provided by Nanjing Xuezi Medical and Chemical Research Center, China; Aβ25-35 was provided by Sigma, USA; pC12 cells were provided by the Institute of Basic Medical Science, Academy of Military Medical Sciences; and rabbit anti-p53 polyclonal antibody was provided by Santa Cruz Biotechnology, USA. METHODS： pC12 cells were cultured in serum-free media for 24 hours. Cells from the model group were treated with 25 μmol/L Aβ25-35 for 24 hours. Cells in the drug protection group were pre-treated with 30 mg/L proanthocyanidins for 1 hour and then treated with 25 μmol/LAβ2^-35 for 24 hours. The control group was not treated. MAIN OUTCOME MEASURES： Flow cytometry was used to detect cell cycle distribution and rate of apoptosis; reverse-transcriptase polymerase chain reaction was used to detect p53 mRNA expression; and Western blot was used to detect p53 protein expression. RESULTS： After treating with 25 μmol/LAβ25-35 for 24 hours, the rate of apoptosis and the percentage of cells in S phase were significantly increased （p 〈 0.01 ）, and p53 mRNA and protein expressions were decreased. p

关键词： proanthocyanidins β-amyloid peptide (25-35) Alzheimer's disease pC12 cells p53 gene neural regeneration

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DETECTION OF p53 gene MUTATION IN pLASMA OF pATIENTS WITH GASTRIC CANCER

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Chinese Journal of Cancer Research 2004年第3期16卷 182-187页

作者：苏鹏程李子禹张连海万文徽任晖张桂国王怡邓国仁季加孚 Department of Surgical Oncology Peking University School of Oncology Beijing Institute for Cancer Research Beijing Cancer Hospital Department of Clinical Laboratory Peking University School of Oncology Beijing Institute for Cancer Research Beijing Cancer Hospital Dapartment of Biochemistry Peking University School of Oncology Beijing Institute for Cancer Research Beijing Cancer Hospital

Objective: To investigated p53 gene mutation in plasma of gastric cancer patients. Methods: DNA extracted from plasma and matched tumor and tumor-adjacent non-cancerous tissues of 96 gastric cancer patients, and DNA from 20 healthy volunteers were studied. Exon 5, 6, 7, and 8 of p53 were amplified by polymerase Chain Reaction (pCR). The mutation status was analyzed by denaturing high-performance liquid chromatography (DHpLC), followed by direct sequencing of cases with aberrant chromatographic patterns. Results: Heterozygous mutations of p53 gene were detected in 19.9% (19/96) of primary tumor tissues and 5.2% (5/96) of corresponding plasma. All p53 gene mutations detected in plasma DNA consisted with mutations in the matched primary tumor samples. Neither the tumor-adjacent gastric mucosa tissues nor control plasma from healthy volunteers showed p53 gene mutation. No correlation was found between p53 mutation status and clinicopathological features of gastric cancer patients. Conclusion: p53 gene mutation in plasma can be detected in tissues and plasma of gastric cancer patients, which could be applied in screening and surveillance of this disease.

关键词： p53 gene Mutation, plasma DNA Gastric cancer

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DETECTION OF p53 gene MUTATION OF BRONCHOSCOpIC SAMpLIES IN THE pATIENTS SUSpECTED TO LUNG CANCER

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Chinese Journal of Cancer Research 2000年第4期12卷 282-285页

作者：郭雪君倪培华李莉邓伟吾万欢英时国朝 Department of Respiratory Medicine Ruijin Hospital Shanghai Second Medical University Shanghai China Department of Medical Laboratory Shanghai Second Medical University Shanghai China

Objective: To determine the feasibility of detecting p53 gene mutations for early diagnosis of lung cancer using the samples from bronchoscopic examination. Methods: point mutations of the exon 5-8 of p53 gene were detected in 85 bronchoscopic samples of 35 patients suspected to be lung cancer using silver staining pCR-SSCp. Results: p53 gene mutations were founded in 10 of 35 patients(28.6%). The incidence of p53 gene mutations (14.9%) was obviously higher than the cytological positive incidence(2.9%) in samples of sputum, bronchoalveolar lavage and brush, especially for the sputum(27.7%). In the bronchoscopic biopsy specimens, the incidence of p53 gene mutations (12.5%) was lower than that of pathologic positive result (50.0%). However, in view of all the bronchoscopic samples, there was no statistically difference between cytopathologic positive results (11.8%) and the incidence of p53 gene mutations (14.1%). Although the p53 mutations were most common in the samples from the patients bronchoscopically manifested as neoplasm compared with other manifestations, there was no statistical difference. It is valuable to notice that 3 patients with p53 gene mutation merely presented as bronchial inflammation in bronchoscope. Conclusion: Results indicated that the value of detecting p53 gene mutation for the diagnosis of lung cancer using the bronchoscopic samples was more superior to cytological examination and detection of p53 gene mutations in post-bronchoscopic sputum was easy and effective, may be used as a valuable method for early diagnosis of lung cancer.

关键词： Lung cancer Bronchoscopy p53 gene Mutation detection

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THE　SIGNIFICANCE　OF　p53　gene　MUTATIONS　AND　EXpRESSIONS　IN　HUMAN　COLORECTAL　TUMORS

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Chinese Journal of Cancer Research 1996年第2期8卷 109-112页

作者：钱桦郁宝铭周锡庚王瑞年黄薇陈赛娟陈竺 Department of Surgery Xinhua Hospital Shanghai Second Medical University Shanghai Department of Surgery Ruijin Hospital SSMU Shanghai Department of pathology SSMU Shanghai Department of Hematology Ruijin Hospital SSMU Shanghai

Using a polymerase chain reaction-single strand conformation polymorphism (pCR-SSCp) approach we analyzed 18 human colorectal adenocarcinomas for mutations in exons 5,6,7,8 of p53 gene. At the same time,p53 gene product expression was studied immunohistochemically in these 18 case in frozen sections. The expression of p53 protein was also immunohistochemically studied in formalin-fixed paraffin embeded spccimens of 76 colorectal adenocarcinomas and 112colorectal polyps. Eigbt out of 18 cases (44%) showed a variant band indicative of a mutation in exons 5-6 of p53gene 7 out of 8 cascs (88%) with p53 gene mutations were positivelystanined for p53. There was no significant correlation between p53. expression and clinicopathological manifestations and prognosis. but the strongest staining was cncountered in those cases with well differentiated and early stage adenocarcinomas,while weaker staining was encountered in poorly differentiated and mucoid adenotarcinomas. p53expression was not observed in proliferative polyps and adenomas with low grade dysplasia. The frequency of p53expression reached 88% (ppes of adenomas, p53 expression was most frequent in villous type (pp53 expression in adenoma, adenoma with malignant change and adenocarcinoma were 4%, 88% and 51% *** indicate that genetic changes of p53 gene play an important role in the transformation from benign adeuoma to adenocarcinoma. p53 immunohistochcmistry can be used as a surrogate marker for p53 gene mutation for early discovery of colorectal adenocarcinomas.

关键词： Colorectal neoplasms p53 gene Mutation,Expression pCR-SSCp

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GROWTH INHIBITION OF HUMAN LARYNGEAL CANCER CELL WITH THE ADENOVIRUS-MEDIATED p53 gene

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Chinese Journal of Cancer Research 1999年第3期11卷 157-160页

作者：王琪韩德民王文革吴祖泽张伟 Department of Otolaryngology-Head and Neck Surgery Institute of Otolaryngology Beijing Tongren Hospital Capital University of Medical Sciences Beijing China

Objective: In most laryngeal cancers, the function of p53 gene is down regulated. To explore the potential use of p53 in gene therapy of laryngeal cancer, by introducing wild-type p53 into laryngeal cancer cell line via a recombinant adenoviral vector, Ad5CMV-p53 and analyzing its effects on cell and tumor growth. Methods: A human laryngeal cancer, cell line Hep-2 was used. Recombinant cytomegalovirus-promoted adenoviruses containing human wild-type p53 cDNA was transiently introduced into Hep-2 line. The growth suppression of the Hep-2 cells and established s.c. squamous, carcinoma model was examined. The p53 protein expression was detected using immunohistochemical analysis. Results: The transduction efficiencies of Hep-2 cell line were 100% at a multiplicity of 100 or greater. The p53 protein expression peaked on day 2 after infection and lasted far 5 days. In vitro growth assays revealed cell death following Ad5CMV-p53 infected. In vivo studies, Ad5CMV-p53 inhibited the tumorigenicity of Hep-2 cell, and in nude mice with established s.c. squamous, carcinoma, nodules showed that tumor volumes were significantly reduced in mice that received peritumoral infiltration of Ad5CMV-p53. Conclusion: Adenovirus-mediated antitumor therapy carrying the p53 gene is an efficient method to inhibit laryngeal cancer growth. Transfection of laryngeal cancer cells with the wild-type p53 gene via Ad5CMV-p53 is a potential novel approach to the therapy of laryngeal cancer.

关键词： gene therapy Laryngeal cancer p53 gene Adenoviruses

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RELATIONSHIp　BETWEEN　THE　MUTATION　OF　p53　gene　AND　INFILTRATION，METASTASIS　AND　pROGNOSIS　OF　GASTRIC　CARCINOMA

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Chinese Journal of Cancer Research 1996年第4期8卷 263-266页

作者：辛彦赵凤凯吴东瑛王艳萍徐蕾张荫昌 Cancer Institute China Medical University Shenyang Department of Histopathology Charring Cross & Westminster Medical School University of London UK

gastric carcinomas were examined immunohistochemically with McAb to p53 protein in order to investigate the relationship between the expression of p53 protein and histological differentiation of gastric carcinoma, and to approach the mechanism of infiltration and metastasis of gastric carcinoma. The results showed that nuclear expression of p53 protein was significantly related to tumor size, depth of invasion, lymph node and liver metastases; but not related to histological differentiation. It is suggested that the accumulation of p53 protein was increased with the progression of gastric carcinoma, and therefore the cancer clone with p53 gene mutation may play an important role in the development of tumor invasion and metastasis.

关键词： p53 gene Tumor suppresser gene.Metastasis Gastric carcinoma

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Effect of Wild-type p53 gene Transfection on the Growth and Radiotherapeutic Sensitivity of Human Glioma Cells

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2005年第4期25卷 448-450页

作者：项炜朱贤立赵洪洋 Department of Neurosurgery Union Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan 430022 China

Summary： To evaluate the effect of wild type p53 gene on the growth and radiotherapeutic sensitivity of human glioma cells, plasmid pC53 SN3 carrying wild type p53 gene was transfected into U251 cells, p53 gene expression in transfected cells was detected by RT pCR, and the cell growth inhibition and apoptosis in the absence or presence of irradiation were assessed by MTT and flow cytometry. The transfection of p53 gene into U251 cells was confirmed by RT pCR. MTT showed that p53 gene alone induced strong inhibitory effect on the growth of U251 cells （inhibition rate （IR）：（79.60±5.69）%）, The killing effect of irradiation alone on U251 cells was not strong （IR：（17. 06±4.35）%, （17.39±1.67）%, （18.73±4.68）%） and increased with the irradiation doses （3, 6, 9 Gy）. When combined treatment of wild-type p53 gene transfection and irradiation was used, the effect was significantly increased （IR：（80.60±5.35）%, （90.30＋1.67） %, （91.30±2.01） %）. Theapoptosis rate of U251 cells induced by p53 gene transfectionwas 17.38 %. The rate induced by irradiation increased （4. 61%, 4. 84%, 5. 40 %） with the irradiation doses （3, 6, 9 Gy）. Theapoptosis rate was also significantly increased （17.80%, 20.03%, 22.34%） after combined treatment of p53 and irradiation with different doses （3, 6, 9 Gy）. It is concluded that wildtype p53 gene and irradiation could result in synergistic inhibitory effect on the growth of human glioma cells.

关键词： gene therapy p53 gene glioma irradiation therapy

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THE RELATIONSHIp OF p53 gene MUTATION AND TUMORINVASION AND LYMpH NODE METASTASIS IN EARLY GASTRIC CANCER

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Journal of pharmaceutical Analysis 1998年第1期12卷 16-21页

作者：徐威车向明

This study was conducted by determination or mutative p53 gene expression in 32 casesof submucosa early gastric cancer. The relationship of p53 gene mutation and tumorlgenesis andprogress or gastric cancer was evaluated based on the cllnlco-pathological characteristics of early gastric cancer. Results showed that positive rate or p53 protein expression was 34. 8% in early gastriccancer and p53 mutation related to the hlstology, location or tumor and lymph node metastasis (p p53 gene ed,resslon closely related to the prognosis of early gastric cancer, and carcinogenesls I,athways may be different according to the positions of stomach.

关键词： p53 gene early gastric cancer pathology lymph node metastasis

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Comparison of Nuclear Accumulation of p53 protein with Mutations in the p53 gene of Human Breast Cancer Tissues

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The Journal of Biomedical Research 2001年第2期15卷 91-95页

作者：范萍武正炎查小明王萱仪 Department of general Surgery the First Affiliated Hospital of Nanjing Medical University

Objective The objective was to compare nuclear accumulation of p53 protein with mutations in the p53 gene on the tissues of human breast cancer. Methods Fifty four invasive ductal carcinomas of breast were analyzed by the method of polymerase chain reaction single strand conformational polymorphism (pCR SSCp) silver stain and strep avidin biotin peroxidase complex (SABC) immunohistochemistry. Results A statistically significant association between the presence of p53 gene mutation and nuclear accumulation of p53 protein was found (pp53 gene mutations showed nuclear accumulation of p53 protein, while only 9 (28%) showed nuclear accumulation of p53 protein in 32 tumors without p53 gene mutations. Both p53 mutation protein and p53 gene mutations were prevalent in steroid and progesterone receptors negative tumors (pp53 protein and lymph node invasion (pp53 gene mutations and lymph node invasion (pp53 abnormalities might be associated with an aggressive phenotype in breast cancer. Conclusion The immunohistochemical detection of nuclear p53 protein accumulation is highly associated with p53 gene mutations in breast cancer tissues, and that this method is useful for rapid screening of p53 abnormalities. However, in order to avoid false positive reaction, the p53 gene mutations should be determined in cases slightly positive for p53 nuclear protein.

关键词： p53 protein p53 gene breast cancer polymerase chain reaction single strand polymorphism

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THE　DETECTION　OF　p53　gene　IN　HUMAN　CERVICAL　CARCINOMA　WITH　AND　WITHOUT　HUMAN　pApILLOMAVIRUS　INFECTION

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Journal of pharmaceutical Analysis 1994年第2期8卷 81-85,101页

作者：孙毅司履生

The special primers Of p53 exon 7 as wed as HpV16 E6 and E7 ORFs (Opening Rending Frame) were used with pCR, pCR-SSCp technique, and 35 specimens of cervical carcinoma were examined. The results were as follows: ① HpV16 E6, E7 DNA was found in 25/35 specimens (71. 4%),which proved again HpV16 Infection an important event in cervical carcinogenesis. However only 11/35 (31.42% ) bad E6 and E7 ORFs simultaneously, 3/35 (8. 57%) and 11/35 (31. 42% ) had only E6 or E7 respectively. ② No mutation and LOH (Loss of Heterozygote) of p53 exon 7 were found in allof 35 specimens. Additionally in the present study, we developed a non-isotopic pCR-SSCp method.

关键词： cervical carcinoma human papillomavirus(HpV) p53 gene polymerase chain reaction (pCR) pCR-single strand comformational polymorphism(SSCp)

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