舞茸Dfraction 通过激活BAK1基因诱导乳腺癌细胞凋亡.docx

1、舞茸Dfraction 通过激活BAK1基因诱导乳腺癌细胞凋亡声明：本文转载自Journal of Medicinal Food ，即药物强化性食品杂志，是由美国Mary Ann Liebert出版公司出版发行的一本有关功能食品及保健食品的国际性杂志。为方便广大读者阅读，我们对论文摘要进行了中文翻译，译文仅供参考，如有不足之处，敬请指正。 -本论文由杭州正树保健食品有限公司整理编辑【参考译文】Maitake (D Fraction) Mushroom Extract Induces Apoptosis in Breast Cancer Cells by BAK-1 Gene Activatio

2、n舞茸D-fraction通过激活BAK-1基因诱导乳腺癌细胞凋亡摘要：根据多年经验，菇类一直被用作传统药物治疗多种疾病。基于对舞茸固有的免疫调节和抗肿瘤特性的研究，人们进一步分离出多种具有生物活性的复合物，其中之一的舞茸D-fraction已被确认具有削弱肿瘤细胞生存能力的功能。本研究验证了舞茸D-fraction对人类乳腺癌细胞（MCF7）生存能力和细胞凋亡的影响。这些细胞分别用浓度为18g/ml, 36g/mL, 91g/m, 183g/mL, 367g/mL的舞茸D-fraction进行处理,另有一组未经处理的细胞作为对照组，实验持续24小时。根据舞茸D-fraction的剂量，处理过

3、的细胞生存能力（根据3-(4,5-二甲基)-5-(3-羧甲基苯环)-2-(4-硫基苯)-2H-四唑盐复合物检测法）依次有所降低。细胞凋亡的统计数依剂量都有明显上升（终端脱氧核糖核酸末端转移酶介导的三磷酸脱氧尿嘧啶缺口端标记法）。细胞经D-fraction恒温培养后，进行微阵列检测，结果显示能直接体现细胞凋亡途径的两种蛋白质BAK-1基因和细胞色素C的转录产物上升。反转录聚合酶链反应也证实了微阵列检查的结果：BAK-1是最过表达的基因之一。这诸多发现更加肯定了舞茸D-fraction对乳腺癌细胞凋亡的影响，并进一步凸显了释放于细胞质的细胞色素C的干预作用。释放在细胞质中的细胞色素C，是细胞凋亡途

4、径中另一个主要因素，其产量在经舞茸D-fraction培养后有所增加，且随D-fraction浓度的增加而上升。这一发现显示，舞茸D-fraction的作用在于诱导线粒体功能障碍。因此，阐明舞茸D-Fraction发挥作用的分子机理对于癌症预防和治疗方法的发展至关重要。关键词：细胞凋亡，BAK-1, 乳腺癌，细胞色素C，舞茸，蘑菇论文原文Journal of Medicinal Food 2011, 1-10Maitake (D Fraction) Mushroom Extract Induces Apoptosisin Breast Cancer Cells by BAK-1 Gene Ac

5、tivationRaquel Soares, Manuela Meireles,1 Ana Rocha,1 Ana Pirraco,1 Diego Obiol,2 Eliana Alonso,2Gisela Joos,2 and Gabriela Balogh21Department of Biochemistry, Faculty of Medicine, University of Porto Foundation, Porto, Portugal.2Center for Scientific and Technical Investigation, Cerzos-Conicet, Bah

6、 Blanca, Argentina.ABSTRACT For many years mushrooms have been used empirically in traditional medicine to treat several diseases. Study of the maitake mushroom, with its immunomodulatory and antitumoral properties, has led to the isolation of several bioactive compounds. One of these, D fraction, i

7、s known to reduce tumor cell viability. This study examined the effect of isolated D fraction on viability and apoptosis of human breast cancer cells (MCF7). These cells were treated with maitake (D fraction) extract at 18g/mL, 36g/mL, 91g/mL, 183g/mL, or 367g/mL or were left untreated (control) for

8、 24 hours. MCF7 incubation with the maitake extract resulted in decreased cell viability 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe- nyl)-2H-tetrazolium assay in a dose-dependent manner. Apoptosis was statistically significantly increased in a dose-dependent manner at ever

9、y concentration tested (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay). Upon incubation with D fraction, a microarray assay revealed upregulation of BAK-1 and cytochrome c transcripts, 2 proteins directly involved in the apoptotic pathway. Reverse t

10、ranscriptase polymerase chain reaction studies confirmed these findings; BAK-1 was one of most overexpressed gene, as observed by microarray assay. These findings confirm the apoptotic effect of maitake D fraction in breast cancer cells and further highlight the involvement of cytochrome c release t

11、o the cytoplasm. Cytoplasmic release of cytochrome c, another player in the apoptotic pathway, was also increased after incubation with D fraction in a dose-dependent manner. This finding indicates that the effect of this compound involves mitochondrial dysfunction. The identification of the molecul

12、ar mechanisms by which D fraction exerts its effects is crucial for the development of preventive and therapeutic strategies for cancer.KEY WORDS: apoptosisBAK-1breast cancercytochrome cmaitakemushroomINTRODUCTION Breast cancer is the leading cause of death from cancer in women, the most common canc

13、er among women worldwide, and the second most frequent cancer in the world. Its incidence is still increasing, with a total number of cases of 1,050,100 in 2002 compared with 572,100 in 1980. Breast cancer causes 370,000 deaths annually, representing 13.9% of cancer deaths in women, mainly in the in

14、dustrialized countries.1 While researchers search for the cause of the disease, an effective and rapid treatment is mandatory for these patients. Maitake mushroom (Grifola frondosa) is a giant mushroom indigenous to northern Japan that has been used by Japanese herbalists for many years. Most resear

15、ch on maitake has focused on the use of maitake D fraction for treating various malignancies. The bioactive D fraction extracted from maitake is a protein-bound polysaccharide compound and is prepared by a standardized procedure developed by Maitake Products Inc. Among the various maitake fractions

16、that have been prepared, D fraction was found to be the most potent for enhancing the immune system via oral administration or injection (both routes were effective), leading to the highest reduction in the rate of cancer proliferation.2 Maitake D fraction has been reported to exert its antitumor ef

17、fect in tumor-bearing mice by enhancing the immune system through activation of macrophages, T cells, and natural killer cells.3 In a previous study, the combination of immunotherapy with the maitake D fraction and chemotherapy suggested that the D fraction might decrease the size of lung, liver, an

18、d breast tumors in patients with cancer.2,4,5 In 1998, the Food and Drug Administration granted Maitake Products, Inc., an investigational new drug application to conduct a phase II pilot study using maitake D fraction among patients with advanced breast and prostate cancers. These ongoing studies a

19、re evaluating the immune stimulatory effect of D fraction on tumor size, immune assays, clinical symptoms, and patient quality of life. The purpose of the current study was to investigate the direct effect of maitake D fraction on breast cancer MCF7 cells. We found that incubating MCF7 cells with ma

20、itake D fraction significantly decreased cell viability and induced cell death. Genomic analysis using complementary DNA (cDNA) microarrays revealed the upregulation of 22 pro-apoptotic and 7 anti-apoptotic genes compared with untreated MCF7 cells (control). BAK-1 transcript at the messenger RNA lev

21、el was found to be upregulated. After incubation with D fraction, cytoplasmic release of cytochrome c, another player in the apoptotic pathway, was also increased in a dose-dependent manner.MATERIALS AND METHODSBioactive maitake D fraction The bioactive D fraction was extracted from maitake mushroom

22、, corresponding to the protein-bound polysaccharide compound, and was prepared by a standardized procedure developed by Maitake Products, Inc.Cell cultures The human breast cancer MCF7 cell line was obtained from the American Type Culture Collection. MCF7 cells were routinely cultured in Eagle minim

23、al essential medium containing 10% inactivated fetal bovine serum and 1% penicillin/streptomycin. Cell culture media, fetal bovine serum, and penicillin/streptomycin were purchased from Invitrogen Life Technologies. Cells were grown at 37 in a humidified 5% CO2 atmosphere. In agreement with previous

24、 studies, incubations were performed for 24 hours in serum-free conditions.6Labeling and cDNA human microarrays We adopted the direct labeling of probes with amine-modified random primer, using 5g of amplified RNA (aaRNA) as starting material. The 5g of aaRNA (8L) was combined with amine-modified ra

25、ndom primer (2g/L,1L) and ribonuclease inhibitor (5 units/L, 1L). The mix was incubated at 70 for 10 minutes and then chilled on ice for 10 minutes. Primer-RNA solution was added to the reverse transcriptase mix (5first-strand buffer, 6L; 50 amplified deoxyuridine triphosphate dUTP/dinucleotide trip

26、hosphate 25mM deoxyadenosine triphosphate, deoxyguanosine triphosphate, and deoxycytidine triphosphate; 15mM deoxythymidine triphosphate; and 10mM aminoallyl-dUTP, 0.6L; dithiothreitol, 0.1M, 3L; Superscript II reverse transcriptase Invitrogen/Life Technologies, 2L) and incubated at 42 for 2 hours.

27、The reaction was terminated by adding EDTA (0.5M, 10L), and the RNA was hydrolyzed with NaOH (1M, 10L) at 65 for 30 minutes.Probe purification Probes were cleaned with a QIA quick polymerase chain reaction (PCR) purification kit (Qiagen); the Cy3- and Cy5- labeled products were combined and 30L of w

28、ater was added, followed by 500L of Buffer PB. The samples were applied to QIA quick columns, which were centrifuged at 13,000 rpm for 1 minute, after which the flow through were discarded. To wash the columns, 750L of Buffer PE was added, columns were spun again for 1 minute, and the flow-through w

29、as discarded. The washing step was repeated once more, and columns were spun again to remove residual ethanol. Fresh collection tubes were placed beneath each column, 30L of Buffer EB was added, and tubes were incubated for 1 minute at room temperature. Columns were then centrifuged at 13,000 rpm fo

30、r 1 minute, and the elution step was repeated once. Elutes were partially dried in a vacuum centrifuge and the volumes were adjusted to 23L with water.Hybridization and washing conditions The following were added: 4.5L of 20saline-sodium citrate, 2L of poly(A) (10mg/mL), and 0.6L of 10% (wt/vol) sod

31、ium dodecyl sulfate (SDS). The probes were then denatured at 98 for 3 minutes. The products were pipetted onto arrays, coverslips were applied, and the slideswere placed in a hybridization chamber (Corning). cDNA human microarrays containing 25,000 known genes were incubated in a 42 water bath for 1

32、6 hours and were subsequently washed with 0.5saline-sodium citrate, 0.01% (wt/vol) SDS, followed by 0.06SSC, at room temperaturefor 10 minutes each. Slides were spun for 5 minutes at 800rpm (130 g) at room temperature.Array scanning and analysis Arrays were read with an Affymetrix 428 fluorescent scanner (MWG Technologies) at 10-m resolution and variable photomultiplier tube voltage settings to obtain the maximal signal intensities with less than 1% (wt/vol) probe saturation. The resulting images were analyzed by using ImaGene, version 4.2, and G