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ABSTRACT: A large amount of DNA of high quality is essential for molecular analysis. The amount of DNA in routine paraffin sections is small. Surgical specimens retained in formalin for the long-term (several months) left over from the sampling required for wax embedding can be referred to as "long-term formalin-fixed" specimens, and clearly this material is a rich source of DNA; however, it is difficult to extract. In the current study, we designed a microwave-heating method for DNA extraction from these specimens. We found that the heating procedure achieved greater DNA yields than a common nonheating method used for comparison (DNA contents mean±SD, heating 2.16±0.95 μg/μL vs. common 1.75±0.90 μg/μL, P<0.05). Fluorescence multiplex polymerase chain reaction-capillary electrophoresis successfully detected microsatellite instability (MSI) in the DNA samples derived from this heating procedure (98.4%, 689 of 700 sample tests), at significantly higher levels than from the conventional method (82.3%, 247 of 300 sample tests, P<0.05). We identified 10 (14.3%) MSI with high frequency and 6 (8.6%) MSI with low frequency colorectal cancers. MSI with high frequency cancers showed distinct clinicopathologic features including higher incidence of right-sided location, high histologic grade, mucin-production, and prominent intraepithelial lymphocyte infiltration. We concluded that the microwave-heating method was efficient for DNA isolation from long-term formalin-fixed tissue samples. The successful fluorescence multiplex polymerase chain reaction-capillary electrophoresis analysis in these samples might facilitate MSI detection in clinical practice.
Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry 04/2012; 20(5):512-7. · 1.63 Impact Factor
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ABSTRACT: To address the association between variants and breast cancer, an increasing number of articles on genetic association studies, genome-wide association studies (GWASs), and related meta- and pooled analyses have been published. Such studies have prompted an updated assessment of the associations between gene variants and breast cancer risk. We searched PubMed, Medline, and Web of Science and retrieved a total of 87 meta- and pooled analyses, which addressed the associations between 145 gene variants and breast cancer. Analyses met the following criteria: (1) breast cancer was the outcome, (2) the articles were all published in English, and (3) in the recent published meta- and pooled analyses, the analyses with more subjects were selected. Among the 145 variants, 46 were significantly associated with breast cancer and the other 99 (in 62 genes) were not significantly associated with breast cancer. The summary ORs for the 46 significant associations (P < 0.05) were further assessed by the method of false-positive report probability (FPRP). Our results demonstrated that 10 associations were noteworthy: CASP8 (D302H), CHEK2 (*1100delC), CTLA4 (+49G>A), FGFR2 (rs2981582, rs1219648, and rs2420946), HRAS (rare alleles), IL1B (rs1143627), LSP1 (rs3817198), and MAP3K1 (rs889312). In addition, eight GWASs were identified, in which 25 loci were obtained (14 in nine genes, six near a gene or genes, and five intergenic loci). Of the 25 SNPs, 20 were noteworthy: C6orf97 (rs2046210 and rs3757318), FGFR2 (rs2981579, rs1219648, and rs2981582), LSP1 (rs909116), RNF146 (rs2180341), SLC4A7 (rs4973768), MRPS30 (rs7716600), TOX3 (rs3803662 and rs4784227), ZNF365 (rs10995190), rs889312, rs614367, rs13281615, rs13387042, rs11249433, rs1011970, rs614367, and rs1562430. In summary, in this review of genetic association studies, 31.7% of the gene-variant breast cancer associations were significant, and 21.7% of these significant associations were noteworthy. However, in GWASs, 80% of the significant associations were noteworthy.
Breast Cancer Research and Treatment 03/2011; 127(2):309-24. · 4.43 Impact Factor
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ABSTRACT: In this study, norcanthridin (NCTD)-encapsulated liposomes were modified with a novel murine anti-human CD19 monoclonal antibody 2E8 (2E8-NCTD-liposomes) and the targeting efficiency and specific cytotoxicity of 2E8-NCTD-liposomes to CD19(+) leukemia cells were evaluated. BALB/c mice were injected with 2E8 hybridoma cells to obtain 2E8 monoclonal antibody (mAb). NCTD-liposomes were prepared by using film dispersion method. 2E8 mAbs were linked to NCTD-liposomes using post-incorporation technology. Flow cytometry showed that the targeting efficiency of purified 2E8 mAbs on CD19(+) Nalm-6 cells was 99.93%. The purified 2E8 mAbs were conjugated with NCTD-liposomes to prepare 2E8-NCTD-liposomes whose targeting efficiency on CD19(+) Nalm-6 was also 95.82%. The average size of 2E8-NCTD-liposomes was 118.32 nm in diameter. HPLC showed that the encapsulation efficiency of NCTD was 46.51%. When the molar ratio of 2E8/Mal-PEG(2000)-DSPE reached 1:50, we obtained the liposomes with 9 2E8 molecules per liposome. The targeting efficiency of 2E8-NCTD-liposomes on CD19(+) leukemia cells was significantly higher than that on CD19-leukemia cells. Similarly, the targeting efficiency of the immunoliposomes was also higher than that of the NCTD-liposomes on CD19(+) leukemia cells. Those results were consistent with those observed by laser scanning confocal microscopy. 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay demonstrated that 2E8-NCTD-liposomes specifically killed Nalm-6 cells in a dose- and time-dependent manner. The viability of Nalm-6 cells treated by 2E8-NCTD-liposomes was significantly lower than that of Molt-3 cells and it was also significantly lower than that of Nalm-6 cells treated with the same concentration of NCTD-liposomes or free NCTD. We are led to concluded that 2E8 antigen can serve as a specific targeting molecule of B lineage hematopoietic malignancies for liposome targeting, and 2E8-NCTD-liposomes can be used as a new and effective means for the treatment of B lineage hematopoietic malignancies.
Journal of Huazhong University of Science and Technology 04/2010; 30(2):240-7. · 0.38 Impact Factor
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ABSTRACT: Lymph node metastasis is the major concern that causes death in colorectal cancers. However, biomarkers for cancer metastasis are still lacking. In this study, we applied an LC-MS/MS-based label-free quantitative proteomics approach to compare the differential secretome of a primary cell line SW480 and its lymph node metastatic cell line SW620 from the same colorectal cancer patient. We identified a total of 910 proteins from the conditioned media and 145 differential proteins between SW480 and SW620 (>1.5-fold change). The differential expression pattern of 6 candidate proteins was validated by Western blot analysis. Among them, trefoil factor 3 and growth/differentiation factor 15, two up-regulated proteins in SW620, were further analyzed in a large cohort of clinical tissue and serum samples. Sandwich ELISA assay showed that the serum levels of both proteins were significantly higher in lymph node metastatic colorectal cancers. Receiver operating characteristic curve analysis confirmed that serum trefoil factor 3 and growth/differentiation factor 15 could provide a discriminatory diagnostic test for predicting colorectal cancer metastasis. Immunohistochemical analysis also showed that the overexpression of trefoil factor 3 or growth/differentiation factor 15 in colorectal cancer was associated with lymph node metastatic behavior. This study showed an accurate, sensitive, and robust label-free quantitation approach for differential analysis of cancer secretome. The comparison of the cancer secretome in vitro is a feasible strategy to obtain valuable biomarkers for potential clinical application. Both trefoil factor 3 and growth/differentiation factor 15 could serve as potential biomarkers for the prediction of colorectal cancer metastasis.
Journal of Proteome Research 11/2009; 9(1):545-55. · 5.11 Impact Factor
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Takeru Oyama,
Yasuhiro Yamada,
Kazuya Hata,
Hiroyuki Tomita,
Akihiro Hirata, Hongqiang Sheng,
Akira Hara,
Hitomi Aoki,
Takahiro Kunisada,
Satoshi Yamashita,
Hideki Mori
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ABSTRACT: Apc(Min/+) mouse, a mouse model for human familial adenomatosis polyposis, contains a truncating mutation in the Apc gene and spontaneously develops intestinal tumors. Our previous study revealed two distinct stages of tumorigenesis in the colon of Apc(Min/+) mouse: microadenomas and macroscopic tumors. Microadenomas already have lost their remaining allele of the Apc and all microadenomas show accumulation of beta-catenin, indicating that activation of the canonical Wnt pathway is an initiating event in the tumorigenesis. This study shows that expression of nuclear beta-catenin in macroscopic tumors is further upregulated in comparison with that in microadenomas. Furthermore, transcriptional activity of beta-catenin/T-cell factor (Tcf) signaling, assessed using beta-catenin/Tcf reporter transgenic mice, is higher in the macroscopic tumors than that in microadenomas. In addition, the expression level of Dickkopf-1, which is known to be a negative modifier of the canonical Wnt pathway, was reduced only in colon tumors. These results suggest that activation of beta-catenin/Tcf transcription plays a role not only in the initiation stage but also in the promotion stage of colon carcinogenesis in Apc(Min/+) mice.
Carcinogenesis 04/2008; 29(3):666-72. · 5.70 Impact Factor
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ABSTRACT: Sesame, which has been reported to have preventive effects against various disordered conditions, contains small quantities of lignans and several precursors to them such as sesaminol glucosides (SG). The lignans have the potent antioxidative activity and are suggested to have chemopreventive property. In the present study, we evaluated the modulating effect of SG on the development of colon precancerous lesions, aberrant crypt foci (ACF) and beta-catenin-accumulated crypts (BCAC), in the azoxymethane (AOM)-induced short-term model using male F344 rats. Dietary SG (500 ppm) significantly decreased the incidence of AOM-induced ACF when compared to the control (P<0.01). The incidences of AOM-induced BCAC in the SG-treated groups (250 or 500 ppm) were also significantly lower than that of the control group (P<0.01). Interestingly, administration of 500 ppm SG clearly decreased serum triglyceride level and mRNA expression of intestinal fatty acid-binding protein in the colonic mucosa, as compared to the control. These findings indicate that dietary SG inhibits AOM-induced carcinogenesis and suggest SG as a possible chemopreventive agent.
Cancer Letters 02/2007; 246(1-2):63-8. · 4.24 Impact Factor
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Huilan Zhi,
Yasuhiro Yamada,
Yoshinobu Hirose,
Keizo Kato, Hongqiang Sheng,
Qiao Zheng,
Takeru Oyama,
Nami Asano,
Toshiya Kuno,
Akira Hara,
Hideki Mori
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ABSTRACT: Aberrant crypt foci (ACF) are recognized as preneoplastic lesions for colon cancer, and ACF in rodents are widely used as an intermediate biomarker to predict tumorigenicity in the colon. However, a lack of correlations between the formation of ACF and the development of colonic tumors has been reported in several studies. For example, 2-(carboxyphenyl) retinamide (2-CPR) and genistein were reported to inhibit the carcinogen-induced formation of ACF, whereas both of them were later found to enhance colon tumorigenesis in rats treated with azoxymethane (AOM). Recently, we have identified b-catenin-accumulated crypts (BCAC) in the colon of rats shortly after administration of AOM, and provided evidence that these are independent early lesions of classical ACF, and BCAC might be direct precursors for colon cancers. In the present study, we performed a comparative analysis of the modifying effects of 2-CPR and genistein on 1,2-dimethylhydrazine (DMH)-induced BCAC and ACF in male F344 rats. Dietary administration of 2-CPR (315 ppm) significantly reduced the total number, multiplicity and size of ACF in DMH-exposed colonic mucosa, while genistein (250 ppm) had no significant effects on DMH-induced ACF formation. In contrast, both of 2-CPR and genistein significantly enhanced the multiplicity and size of DMH-induced BCAC when compared with DMH alone group. In addition, both 2-CPR and genistein significantly increased the proliferating cell nuclear antigen (PCNA) index preferentially in BCAC. Together with previous findings that 2-CPR and genistein are tumor promoters in the colon, our results support the concept that BCAC are precursors of colon tumors and suggest that these lesions are more reliable short-term biomarkers for colon carcinogenesis in rodents than ACF.
Asian Pacific journal of cancer prevention: APJCP 8(1):33-8. · 0.66 Impact Factor
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