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Peng Wang,
Jie Ding, Tao Lin,
Shuang Han,
Shan-shan Cao,
Fu-lin Ge,
Guang-qun An,
Rong Li,
Ting Lei,
Fei-hu Bai,
Dai-ming Fan
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ABSTRACT: To investigate the binding effect of the short peptide SY1 to the multidrug-resistant gastric cancer cell line SGC7901/VCR cells and its reversing effect on those cancer cells.
The cultured cells were divided into two groups named SGC7901 and SGC7901/VCR. The SGC7901/VCR group was co-cultured with vincristine (VCR). SY1 was obtained from cyclic 7-mer peptide library by differential screening. Immunofluorescence technique was used to detect the capacity of SY1-containing positive phage specifically binding to SGC7901/VCR cells, compared with that of the negative phage and unrelated phage. MTT assay in vitro was performed to analyze the alteration of drug resistance of SGC7901/ VCR cells, using the positive phages and the chemically synthesized SY1 peptide. Flow cytometry assay was performed to detect the accumulation and retention of adriamycin (ADM) in the SGC7901/VCR cells.
Immunofluorescence analysis showed that the SY1-containing positive phages could bind to the SGC7901/VCR cell surface but not to its parent cell line SGC7901 cells. The unrelated phage and negative phage did not bind to SGC7901/VCR cells. These results indicated that SY1 could specifically bind to SGC7901/VCR cells. MTT assay in vitro showed that the survival rate of SGC7901/VCR cells was reduced considerably by the positive phages and the chemically synthesized SY1 peptide (P <0. 05), indicating that SY1 enhanced the sensitivity of SGC7901/VCR cells to chemotherapeutic drug VCR. Flow-cytometric detection showed that SY1 enhanced the accumulation of ADM in the SGC7901/VCR cells, compared with that of the negative phages and the unrelated phages (P <0.05).
SY1 not only is able to bind to SGC7901/VCR cells specifically, but also can partly reverse the resistance of SGC7901/VCR cell line to chemotherapeutic drug VCR. Those findings might be important to open a new approach to reverse the gastric cancer MDR.
Zhonghua zhong liu za zhi [Chinese journal of oncology] 05/2007; 29(4):258-61.
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Jun Wang,
Kaichun Wu,
Feihu Bai,
Huihong Zhai,
Huahong Xie,
Yulei Du,
Jie Liang,
Shuang Han,
Yu Chen, Tao Lin,
Daiming Fan
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ABSTRACT: We investigated the potential effect of Cyclooxygenase-2 (Cox-2) on hypoxia-induced Angiopoietin-2 (Ang-2) expression in gastric cancer cells. Our results revealed that hypoxia augmented Cox-2 and Ang-2 expressions. Also, the hypoxia-induced Ang-2 could be mimicked by CoCl(2) treatment while genestein treatment could partially counteract the hypoxia-induced Ang-2 expression. Celecoxib but not Cox-1 inhibitor sc-560 reversed the hypoxia-induced Ang-2 expression, while this effect could be partially restored by addition of exogenous PGE2. Our findings suggest that the hypoxia-elevated Ang-2 expression in gastric cancer cells may be mediated by both Cox-2-derived PGE2 and HIF-1alpha pathways, while celecoxib could counteract the hypoxia-induced Ang-2 expression.
Cancer Letters 11/2006; 242(1):20-7. · 4.24 Impact Factor
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Shuhui Liang, Tao Lin,
Jie Ding,
Yanglin Pan,
Dongmei Dang,
Changcun Guo,
Min Zhi,
Pengtao Zhao,
Li Sun,
Liu Hong,
Yongquan Shi,
Liping Yao,
Jie Liu,
Kaichun Wu,
Daiming Fan
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ABSTRACT: Antiangiogenesis therapy has become a hot field in cancer research. Blood vessels of tumor carry specific markers that are usually related to angiogenesis. Study of these heterogeneous molecules in different tumor vessels may be beneficial for promoting antiangiogenic therapy. In this study, we established an in vitro co-culture model of human umbilical vein endothelial cells (HUVECs) and gastric adenocarcinoma cell line SGC7901, screened the peptides binding specifically to the HUVECs co-cultured with gastric cancer cells (Co-HUVECs) using phage display peptides library, and studied the affinity of these peptides to gastric cancer vascular endothelial cells. After four rounds of panning, there was an obvious enrichment for the phages specifically binding to the Co-HUVECs, and the output/input ratio of Co-HUVECs increased about 590-fold (from 0.95x10(-7) to 561.25x10(-7)). Five phage clones (M6, M3, M9, IN12, IN11), which could strongly bind to Co-HUVECs instead of wild-type HUVECs, were characterized by ELISA. In vitro cellular binding assay showed that phage IN11 preferably bound to Co-HUVECs rather than control HUVECs, and the number of the phage IN11 recovered from Co-HUVECs was 5.7- and 16.9-folds, respectively, as much as those from control HUVECs and GES cells. Immunocytochemical and immunohistochemical staining confirmed that phage IN11 could specifically bind to Co-HUVECs as well as vascular endothelial cells in gastric cancer tissue sections. Competitive and inhibitory assay revealed the synthetic peptide GEBP11 (CTKNSYLMC) displayed on phage IN11 could competitively inhibit binding of the phage IN11 to Co-HUVECs. Immunofluorescence microscopy showed that the fluorescence-labeled peptide GEBP11 was located at the membrane and perinuclear cytoplasm of Co-HUVECs. Meanwhile, GEBP11 was found to be able to target the gastric cancer vascular endothelial cells. Therefore, GEBP11 may be a potential candidate for targeted drug delivery in antivascular therapy and diagnosis of gastric cancer.
Journal of Molecular Medicine 10/2006; 84(9):764-73. · 4.67 Impact Factor
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ABSTRACT: We previously showed that downregulation of a transcription-associated gene (ZNRD1) could reverse the resistant phenotype of gastric cancer cells through regulation of the transcription of multidrug resistance gene 1 (MDR1). In the present study, we determined both known and novel differentially expressed genes in VCR-induced multidrug resistant gastric cancer cell SGC7901/VCR transfected with ZNRD1 siRNA or empty vector control. Screening was performed using the Human Cancer Xpro(tm) HC-III plus arrays, containing 3072 cancer-related cDNAs. Ten genes, involved in cell cycle control, nucleic acid binding, and protein phosphorylation, among other functions, underwent more than 5-fold change. Of the downregulated genes we chose Inosine monophosphate dehydrogenase 2 (IMPDH2) for further validation by quantitative RT-PCR. In vitro and in vivo drug sensitivity analyses revealed that inhibition of ZNRD1 and IMPDH2 activity sensitized SGC7901/VCR cells to methotrexate. Additionally, inhibition of ZNRD1 could suppress adriamycin-induced apoptosis and significantly downregulate the expression of Bcl-2, but it did not alter the expression of the glutathione-S-transferase, or intracellular glutathione content. Taken together, the findings suggest that ZNRD1 could act as a modulator of methotrexate chemotherapy in gastric cancer cells through the regulation of IMPDH2 and Bcl-2.
Biochemistry and Cell Biology 05/2006; 84(2):199-206. · 2.67 Impact Factor
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ABSTRACT: Previously, a novel protein, MGr1-Ag, was associated with tumor multidrug resistance (MDR), and the role and the underlying mechanisms of MGr1-Ag in MDR of gastric cancer cells were characterized. Initial studies using the introduction of sense or antisense vectors for MGr1-Ag resulted in the genetical up- or downregulation of MGr1-Ag in gastric cancer cells, respectively. Subsequent studies revealed the expression of MGr1-Ag, P-glycoprotein (P-gp), MDR-associated protein (MRP), Bcl-2 and Bax in gastric cancer cells via Western blot analysis. The sensitivity of gastric cancer cells to chemotherapeutic drugs was assessed using the colony-forming assay, and Adriamycin (ADM) accumulation was evaluated by flow cytometry. Further study of ADM-induced apoptosis was detected by annexin-V/propidium iodide staining. The expression level of MGr1-Ag in MDR gastric cancer cells is much higher than that in their parental cells. Overexpression of exogenous MGr1-Ag may promote the MDR phenotype of gastric cancer cells, decrease intracellular ADM accumulation and protect gastric cancer cells from ADM-induced apoptosis, whereas downregulation of MGr1-Ag had reverse effects. Western blot analysis suggested that MGr1-Ag may regulate the expression of P-gp, MRP, Bcl-2 and Bax. In conclusion, MGr1-Ag may promote MDR of gastric cancer cells via a decrease in intracellular drug accumulation and inhibition of drug-induced apoptosis.
Tumor Biology 02/2006; 27(1):27-35. · 1.94 Impact Factor
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ABSTRACT: The Runx3 gene is a member of the runt domain family transcription factors, key regulators of development and differentiation in metazoan. Recently, Runx3 was identified as a tumor suppressor gene. Loss of Runx3 was found to be associated with genesis and progression of gastric cancer. In this study, we transfected the gastric cancer cell line SGC7901 with eukaryotic expression vector of Runx3. In vitro drug sensitivity assay suggested that SGC7901/Runx3 cells were more sensitive to various chemotherapeutic drugs. Blocking Runx3 expression in immortalized stomach mucosal cells (GES-1) or gastric cancer cells (SGC7901) by Runx3-specific small interfering RNA conferred the cells resistance to chemotherapeutic drugs. Flow cytometry examination suggested that expression of Runx3 in gastric cancer cells increased the intracellular accumulation and retention of adriamycin. Semiquantitative RT-PCR and Western blot suggested that Runx3 downregulated expression of Bcl-2, MDR-1 (P-gp) and MRP-1. Binding of Runx3 to promoter sequences of Bcl-2, MDR-1 and MRP-1 gene was detected by eletrophoretic mobility shift assay (EMSA) and supershift EMSA. We cloned the MDR-1 and MRP-1 gene promoters containing Runx binding sites and constructed the luciferase reporter vectors of these 2 promoters. Luciferase reporter assay suggested that Runx3 inhibited the promoter activity of the MDR-1 and MRP-1 promoter in SGC7901 cells. Taken together, our findings suggested that overexpression of Runx3 could sensitize gastric cancer cells to chemotherapeutic drugs by downregulating the Bcl-2, MDR-1 and MRP-1.
International Journal of Cancer 09/2005; 116(1):155-60. · 5.44 Impact Factor
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ABSTRACT: Gastric cancer is one of the most common malignant tumors in China. This paper focuses on the development of a DNA vaccine containing four mimotopes of MG7Ag for gastric cancer (multi-epitope vaccine). By inoculating BALB/c mice, the vaccine was characterized and compared with a similar vaccine containing only one mimotope (mono-epitope vaccine) and other controls. Cellular ELISA indicated that serum titer of antibody against MG7Ag was significantly higher in mice immunized with the multi-epitope vaccine than that in the group immunized with the mono-epitope vaccine (0.8627 vs 0.6754, P < 0.05). And ELISPOT assay showed that the number of INF-gamma spots induced by multi-epitope vaccine was significantly larger than that of the group immunized with mono-epitope vaccine(93.3 vs 70.7, P < 0.05). Two weeks after tumor challenge, the weight of tumor in each mouse was evaluated, and the tumor masses formed in the mice immunized with multi-epitope vaccine were markedly smaller than those formed in the mice immunized with mono-epitope vaccine. These studies demonstrated that both humoral and cellular response were induced by the two vaccines and the efficiency of multi-epitope vaccine is stronger than that of the mono-epitope vaccine.
Cancer biology & therapy 03/2005; 4(3):308-12. · 2.64 Impact Factor
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ABSTRACT: CpG oligodeoxynucleotides (CpG ODN) have been shown to have potent adjuvant activity for a wide range of antigens. Of particular interest is their improved activity when closely associated with the antigen. The purpose of this study is to construct a nanovaccine coencapsulated with a gastric cancer specific antigen MG7 mimotope peptide and adjuvant CpG ODN 1645 using new nanotechnology as nanoemulsion and evaluate its immunocompetence. Nanoemulsion vaccine was prepared using magnetic ultrasound methods. BALB/c mice were immunized and the in vivo effectiveness was evaluated using tumor challenge assay. It was shown that the tumor masses formed in the mice immunized with coencapsulated nanovaccine (0.0825 g) markedly smaller (P < 0.01) than those formed in the mice immunized with nanovaccine encapsulated with antigen peptide alone (0.4465 g). A tumor inhibiting rate as high as 82.5% of the coencapsulated nanovaccine was obtained, while nanovaccine encapsulated with peptide only could not achieve the same effect (28.5%) (P < 0.01). Enzyme-linked immunospot assay (ELISPOT) showed that immunization using MG7 mimotope peptide coencapsulated with CpG ODN within the same nanoemulsion enhanced the frequency of splenocytes secreting IFN-gamma significantly (P < 0.01) when compared with immunization using MG7 peptide encapsulated in nanoemulsion alone (197spots/1 x 10(6) vs. 73 spots/1 x 10(6)). Cellular ELISA indicated that serum titer of antibody against MG7-Ag was significantly higher (P < 0.01) in mice immunized with coencapsulation form nanovaccine (0.7884) than that in the group immunized with nanovaccine encapsulated with MG7 peptide alone (0.3616). Using intracellular flow cytometric analysis, it was found that the IFN-gamma response was contributed by CD4+ T-cells. Our experiments suggest that a vaccinal approach using nano-delivery system carrying in tumoral epitope and CpG ODN as adjuvant may have important implications for cancer therapy.
Cancer biology & therapy 02/2005; 4(2):218-24. · 2.64 Impact Factor
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Zhe-Yi Han,
Kai-Chun Wu,
Feng-Tian He,
Quan-Li Han,
Yong-Zhan Nie,
Ying Han,
Xiao-Nan Liu,
Jian-Yong Zheng,
Mei-Hong Xu, Tao Lin,
Dai-Ming Fan
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ABSTRACT: Using a monoclonal antibody against gastric cancer antigen named MGb1 to screen a phage-displayed random peptide library fused with coat protein pIII in order to get some information on mimotopes.
Through affinity enrichment and ELISA screening, positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced) By blocking test, specificity of the mimic phage epitopes was identified.
There were approximately 200 times of enrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGb1-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced. According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGb1-Ab to gastric cancer cell KATO-III. The percentage of blocking was from (21.0+/-1.6) % to (39.0+/-2.7) %.
Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.
World Journal of Gastroenterology 10/2003; 9(9):1920-4. · 2.47 Impact Factor
