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Dai-Ming Fan

Publications and source records attributed to Dai-Ming Fan.

35 records · Page 2Linked to original sources
PubMed2003

[Screening of bioactive peptide that mimic the epitope of gastric cancer associated antigen].

AIM: To screen bioactive peptides that mimic the epitope of gastric cancer associated antigen. METHODS: Anti-gastric cancer monoclonal antibody (mAb) MG7 was purified by ion chromatography, and then coated on ELISA plate. By using the mAb as selective molecular, a 12-meres phage displaying peptide library was biopanned, and the positive clones were selected. RESULTS: 12 positive phage clones were selected. CONCLUSION: A candidate mimic epitope of gastric cancer associated antigen was identified, which provided the foundation for developing tumor peptide vaccine.

Bacteriophages↗
PubMed2002

Direct effect of croton oil on intestinal epithelial cells and colonic smooth muscle cells.

AIM: To investigate the direct effect of croton oil (CO) on human intestinal epithelial cell (HIEC) and guinea pig colonic smooth muscle cells in vitro. METHODS: Growth curves of HIEC were drawn by MTT colorimetry. The dynamics of cell proliferation was analyzed with flow cytometry, and morphological changes were observed under light and electron microscopy after long-term (6 weeks) treatment with CO. Expression of cyclooxygenase-2 (COX-2) mRNA was detected by dot blot in HIEC treated with CO. Genes related to CO were screened by DD-PCR, and the direct effect of CO on the contractility of isolated guinea pig colonic smooth muscle cells was observed. RESULTS: High concentration (20-40 mg x L(-1)) CO inhibited cell growth significantly (1, 3, 5, 7d OD sequence: (20 mg x L(-1)) 0.040+/-0.003, 0.081+/-0.012, 0.147+/-0.022,0.024+/-0.016; (40 mg x L(-1)) 0.033+/-0.044, 0.056+/-0.012, 0.104+/-0.010, 0.189+/-0.006; OD control 0.031+/-0.008, 0.096+/-0.012, 0.173+/-0.009, 0.300+/-0.016, P<0.01), which appeared to be related directly to the dosage. Compared with the control, the fraction number of cells in G1 phase decreased from 0.60 to 0.58, while that in S phase increased from 0.30 to 0.34 and DNA index also increased after 6 weeks of treatment with CO (the dosage was increased gradually from 4 to 40 mg x L(-1)). Light microscopic observation revealed that cells had karyomegaly, less plasma and karyoplasm lopsidedness. Electron microscopy also showed an increase in cell proliferation and in the quantity of abnormal nuclei with pathologic mitosis. Expression of COX-2 mRNA decreased significantly in HIEC treated with CO. Thirteen differential cDNA fragments were cloned from HIEC treated with CO, one of which was 100 percent homologous with human mitochondrial cytochrome C oxidase subunit II. The length of isolated guinea pig colonic smooth muscle cells was significantly shortened after treatment with CO (P<0.05). CONCLUSION: At a high CO concentration (>20 mg x L(-1)), cell growth and proliferation are inhibited in a dosage-dependent manner. Increase in cell proliferation and in malignant conversion of the cellular phenotype is observed in cells cultured chronically with CO. COX-2 mRNA expression decreases significantly, while human mitochondrial cytochrome C oxidase subunit IImRNA expression increases significantly in HIEC treated with CO. CO also has a direct effect on the contractility of Guinea pig colonic smooth muscle cells.

Animals↗
PubMed2002

Differential display of vincristine-resistance-related genes in gastric cancer SGC7901 cell.

AIM: To isolate and clone the vincristine-resistance-related genes in gastric cancer SGC7901 cell line and to clarify the multidrug-resistant molecular mechanism of gastric cancer cells. METHODS: The modified differential-display polymerase chain reaction (DD-PCR) was used to examine differences in the mRNA composition of Vincristine-resistant gastric cancer SGC 7901 cells (SGC7901/VCR), induced by vincristine sulfate versus SGC7901cells. The differentially expressed cDNA fragments were confirmed by reverse Northern analysis, sequencing, BLAST analysis and Northern bolt analysis. RESULTS: The DD-PCR identified that 54 cDNA fragments were preferentially expressed in SGC 7901/VCR cells. When these cDNA fragments were analyzed by reverse Northern blot, twenty were reproducibly expressed at high level in SGC7901/VCR. Sequencing and BLAST analysis revealed that seven of the genes were known genes:ADP-ribosylation factor 4, Cytochrome oxidase subunit II, Ss-A/Ro ribonucleoprtein autoantigen 60kd subunit,ribosomal protein S13, galaectin-8 gene, oligophrenin 1 mRNA, ribosomal protein L23 mRNA; thirteen of the genes were unknown genes. The length and abundance of the four unknown genes were further confirmed by Northern blot analysis. CONCLUSION: The twenty differential known and unknown genes may be related to the vincristine-resistant mechanism in human gastric cancer SGC7901 cell line.

ADP-Ribosylation Factors↗
PubMed2002

Expression and bioactivity identification of soluble MG7 scFv.

AIM: To examine the molecular mass and identify the bioactivity of MG7 scFv for its application as a targeting mediator in gene therapy of gastric cancer. METHODS: Two strongly positive recombinant phage clones screened from MG7 recombinant phage antibody library were separately transfected into E.coli TG1. Plasmid was isolated from the transfected E.coli TG1 and digested by EcoR I and Hind III to examine the length of exogenous scFv gene. Then, the positive recombinant phage clones were individually transfected into E.coli HB2151. The transfectant was cultured and induced by IPTG. Perplasmic extracts was prepared from the induced transfectant by osmotic shock. ELISA was used to examine the antigen-binding affinity of the soluble MG7 scFv. Immunodotting assay was adopted to evaluate the yield of soluble MG7 scFv produced by transfected E.coli HB2151. Western blot was used to examine the molecular mass of MG7 scFv. Finally, the nucleotide sequence of MG7 scFv was examined by DNA sequencing. RESULTS: Two positive recombinant phage clones were found to contain the exogenous scFv gene. ELISA showed that MG7 scFv had strong antigen-binding affinity. Immuodotting assay showed that transfected E.coli HB2151 could successfully produce the soluble MG7 scFv with high yield via induction by IPTG. The molecular mass of MG7 scFv was 30 kDa by western blot. DNA sequencing demonstrated that the VH and VL genes of MG7 scFv were 363 bp and 321 bp,respectively. CONCLUSION: We have successfully developed the soluble MG7 scFv which possessed strong antigen-binding affinity.

Antibody Affinity↗
PubMed2002

Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3.

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.

Animals↗
PubMed2002

Effect of L-NAME on nitric oxide and gastrointestinal motility alterations in cirrhotic rats.

AIM: To investigate the effect of L-NAME on nitric oxide and gastrointestinal motility alterations in cirrhotic rats. METHODS: Rats with cirrhosis induced by carbon tetrachloride were randomly divided into two groups, one n =13 receiving 0.5mg.kg(-1) per day of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, for 10 days, whereas the other group (n =13) and control (n =10) rats were administrated the same volume of 9g.L(-1) saline. Half gastric emptying time and 2h residual rate were measured by SPECT, using (99m)Tc-DTPA-labeled barium sulfate as test meal. Gastrointestinal transition time was recorded simultaneously. Serum concentration of nitric oxide (NO) was determined by the kinetic cadmium reduction and colorimetric methods. Immunohistochemical SABC method was used to observe the expression and distribution of three types of nitric oxide synthase (NOS) isoforms in the rat gastrointestinal tract. Western blot was used to detect expression of gastrointestinal NOS isoforms. RESULTS: Half gastric emptying time and trans-gastrointestinal time were significantly prolonged(124.0 +/- 26.4 min; 33.7 +/- 8.9 min; 72.1 +/- 15.3 min; P<0.01), (12.4 +/- 0.5h; 9.5 +/- 0.3h; 8.2 +/- 0.8h; P<0.01), 2h residual rate was raised in cirrhotic rats than in controls and cirrhotic rats treated with L-NAME (54.9 +/- 7.6%,13.7 +/- 3.2%, 34.9 +/- 10.3%, P<0.01). Serum concentration of NO was significantly increased in cirrhotic rats than in the other groups (8.20 +/- 2.48) micromol.L(-1), (5.94 +/-1.07) micromol.L(-1) and control (5.66 +/- 1.60 micromol.L(-1), P<0.01. NOS staining intensities which were mainly located in the gastrointestinal tissues were markedly lower in cirrhotic rats than in the controls and cirrhotic rats after treated with L-NAME. CONCLUSION: Gastrointestinal motility was remarkably inhibited in cirrhotic rats, which could be alleviated by L-NAME. Nitric oxide may play an important role in the inhibition of gastrointestinal motility in cirrhotic rats.

Animals↗
PubMed2002

Expression and function of classical protein kinase C isoenzymes in gastric cancer cell line and its drug-resistant sublines.

AIM: To investigate the expression and function of classical protein kinase C (PKC) isoenzymes in inducing MDR phenotype in gastric cancer cells. METHODS: Two cell lines were used in the study: gastric cancer cell SGC7901 and its drug-resistant cell SGC7901/VCR stepwise-selected by vincristine 0.3, 0.7 and 1.0 mg.L(-1), respectively. The expression of classical PKC (cPKC) isoenzymes in SGC7901 cells and SGC7901/VCR cells were detected using immunofluorescent cytochemistry, laser confocal scanning microscope and Western blot. The effects of anti-PKC isoenzymes antibody on adriamycin accumulation in SGC7901/VCR cells were determined using flow cytometric analysis. RESULTS: (1)SGC7901 cells exhibited positive staining of PKC-alpha. SGC7901/VCR cells exhibited stronger staining of PKC-alpha than SGC7901 cells. The higher dosage vincristine selected, the much stronger staining of PKC-alpha was observed on SGC7901/VCR cells. (2)Both SGC7901 and SGC7901/VCR cells exhibited positive staining of PKC-beta I and PKC-beta II with no significant difference. (3) Compared with SGC7901, SGC7901/VCR cells had decreased adriamycin accumulation and retention. Accumulation of adriamycin in SGC7901 was 5.21+/-2.56 mg.L(-1),in SGC7901/VCR 0.3 was 0.85+/-0.29 mg.L(-1), in SGC7901/VCR 0.7 was 0.81+/-0.32 mg.L(-1), and in SGC7901/VCR 1.0 was 0.80+/-0.33 mg.L(-1); Retention of adriamycin in SGC 7901 was 2.51+/-1.23 mg.L(-1), in SGC7901/VCR 0.3 was 0.47+/-0.14 mg.L(-1), in SGC7901/VCR 0.7 was 0.44+/-0.15 mg.L(-1), and in SGC 7901/VCR 1.0 was 0.41+/-0.11 mg.L(-1). (4) Fluorescence intensity presented adriamycin accumulation in SGC7901/VCR cells was increased from 1.14+/-0.36 to 2.71+/-0.94 when cells were co-incubated with anti-PKC-alpha but not with anti-PKC-beta I PKC-beta II and PKCgamma antibodies. CONCLUSION: PKC-alpha, but not PKC-beta I, PKC-beta II or PKCgamma, may play a role in multidrug resistance of gastric cancer cells SGC7901/VCR.

Antineoplastic Agents, Phytogenic↗
PubMed2002

Identification of tumor associated single-chain Fv by panning and screening antibody phage library using tumor cells.

AIM: To study the feasibility of panning and screening phage-displaying recombinant single-chain variable fragment (ScFv) of anti-tumor monoclonal antibodies for fixed whole cells as the carriers of mAb-binding antigens. METHODS: The recombinant phage displaying libraries for anti-colorectal tumor mAb MC3Ab, MC5Ab and anti-gastric tumor mAb MGD1 was constructed. Panning and screening were carried out by means of modified fixation of colorectal and gastric tumor cells expressed the mAb-binding antigens. Concordance of binding specificity to tumor cells between phage clones and parent antibodies was analyzed. The phage of positive clones was identified with competitive ELISA, and infected by E.coli HB2151 to express soluble ScFv. RESULTS: The ratio of positive clones to MC3-ScF-MC5-ScFv and MGD1-ScFv were 60 %, 24 % and 30 %. MC3-ScFv had M(r) 32 000 confirmed by Western blot. The specificity to antigen had no difference between 4 positive recombinant phage antibodies and MC3Ab. CONCLUSION: The modified process of fixing whole tumor cells is efficient, convenient and feasible to pan and screen the phage-displaying ScFv of anti-tumor monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed2000

Suppression of P-gp induced multiple drug resistance in a drug resistant gastric cancer cell line by overexpression of Fas.

AIM:To observe the drug sensitizing effect and related mechanisms of fas gene transduction on human drug-resistant gastric cancer cell SGC7901/VCR (resistant to Vincristine).METHODS:The cell cycle alteration was observed by FACS. The sensitivity of gastric cancer cells to apoptosis was determined by in vitro apoptosis assay. The drug sensitization of cells to several anti-tumor drugs was observed by MTT assay. Immunochemical method was used to show expression of P-gp and Topo II in gastric cancer cells.RESULTS:Comparing to SGC7901 and pBK-SGC7901/VCR, fas-SGC7901/VCR showed decreasing G2 cells and increasing S cells, the G2 phase fraction of pBK-SGC7901/VCR was about 3.0 times that of fas -SGC7901/VCR but S phase fraction of fas -SGC7901/VCR was about 1.9 times that of pBK-SGC7901/VCR, indicating S phase arrest of fas-SGC7901/VCR. FACS also suggested apoptosis of fas-SGC7901/VCR.fas-SGC7901/VCR was more sensitive to apoptosis inducing agent VM-26 than pBK-SGC7901/VCR. MTT assay showed increased sensitization of fas-SGC7901/VCR to DDP, MMC and 5-FU, but same sensitization to VCR according to pBK-SGC7901/VCR. SGC7901, PBK-SGC7901/VCR and fas -SGC7901/VCR had positively stained Topo II equally. P-gp staining in pBK-SGC7901/VCR was stronger than in SGC7901, but there was little staining of P-gp in fas-SGC7901/VCR.CONCLUSION:fas gene transduction could reverse the MDR of human drug-resistant gastric cancer cell SGC7901/VCR to a degree, possibly because of higher sensitization to apoptosis and decreased expression of P-gp.

Journal Article↗
PubMed1999

Antisense to cyclin D1 reverses the transformed phenotype of human gastric cancer cells.

AIM:To further investigate the effect of cyclin D1 on the biologic behavior of cancer cells and its potential role in gene therapy of tumor.METHODS:A cyclin D1 subcloning plasmid termed BKSD1 was constructed by subcloning the human cyclin D1 cDNA into Bluescript-KS, a plasmid vector with a pair of T7 and T3 promoters, with recombinant DNA technology of molecular biology. So,it is easy to generate digoxigenin (DIG)-labeled RNA probes of antisense and sense to cyclin D1 using RKSD1 as a template vector. PDORD1AS, an eukaryotic expression vector containing the full-length human cyclin D1 cDNA in its antisense orientation cloned into the retroviral vector pDOR-neo, was successfully constructed with BKSD1 to change restriction sites. A gastric cancer cell line, SGC7901/VCR, was transfected with pDORD1AS by Lipofect Amine-mediated introduction and a subline termed SGC7901/VCRD1AS, which had stable overexpression of antisense RNA to cyclin D1, was obtained by selection in G418. The subline, control subline transfected pDOR-neo and SGC7901/VCR were evaluated by methods of immunohistochemistry, flow cytometry, molecular hybridization, morphology and cell biology.RESULTS:Compared with control cell lines, SGC7901/VCRD1AS had a reduced expression of cyclin D1 (inhibition rate was about 36%), increased cell size and cytoplasm to nucleus ratio, increased doubling time (42.2h to 26.8h and 26.4h), decreased saturation density (18.9X10(4) to 4.8X10(5) and 4.8X10(5)), increased percentage of cells in the G(1)/G(0) phase (80.9%-64.6% and 63.8%), reacquired serum dependence, and a loss of tumorigenicity in nude mice (0/4 to 4/4 and 4/4).CONCLUSION: Stable overexpression of antisense RNA to cyclin D1 can reverse the transformed phenotype of human gastric cancer cells and may provide an approach of gene therapy for gastric cancer.

Journal Article↗
PubMed1999

Down-regulation of Hsp90 could change cell cycle distribution and increase drug sensitivity of tumor cells.

AIM:To construct Hsp90 antisense RNA eukaryotic expression vector, transfect it into SGC7901 and SGC7901/VCR of MDR-type human gastric cancer cell lines, HCC7402 of human hepatic cancer and Ec109 of human esophageal cancer cell lines, and to study the cell cycle distribution of the gene transected cells and their response to chemotherapeutic drugs.METHODS:A 1.03kb cDNA sequence of Hsp90beta was obtained from the primary plasmid phHSP90 by EcoR I and BamH I nuclease digestion and was cloned to the EcoR I and BamH I site of the pcDNA by T4DNA ligase and an antisense orientation of Hsp90beta expression vector was constructed. The constructs were transfected with lipofectamine and positive clones were selected with G418. The expression of RNA was determined with dot blotting and RNase protection assay, and the expression of Hsp90 protein determined with western blot. Cell cycle distribution of the transfectants was analyzed with flow cytometry, and the drug sensitivity of the transfectants to Adriamycin (ADR), vincrinstine (VCR), mitomycin (MMC) and cyclophosphamide (CTX) with MTT and intracellular drug concentration of the transfectants was determined with flow cytometry.RESULTS:In EcoR I and BamH I restriction analysis, the size and the direction of the cloned sequence of Hsp90beta remained what had been designed and the gene constructs were named pcDNA-Hsp90.AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones all expressed Hsp90 anti-sense RNA. The expression of Hsp90 was down-regulated in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones. Cell cycle distribution was changed differently. In AH-SGC7901/VCR and AH-Ec109 cells, G(1) phase cells were increased; S phase and G(2) phase cells were decreased as compared with their parental cell lines. In AH-SGC7901 cell, G(1)phase cells were decreased, G(2) phase cells increased and S phase cells were not changed, and in AH-HCC7402 cells G(1), S and G(2) phase cells remained unchanged as compared with their parental cell lines. The sensitivity of AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 to chemotherapeutic drugs, the sensitivity of AH-SGC7901/VCR to ADR, VCR, MMC and CTX the sensitivity of AH-HCC7402 to ADR and VCR, and the sensitivity of Ec109 to ADR, VCR and CTX all increased as compared with their parental cell lines. The mean fluorescence intensity of ADR in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 was also significantly elevated (P < 0.05).CONCLUSION: Down-regulation of Hsp90 could change cell cycle distribution and increase the drug sensitivity of tumor cells.

Journal Article↗
PubMed1999

Cloning and identification of an angiostatic molecule IP10/crg -2.

AIM:To obtain human and murine cDNAs encoding IFN-gamma inducible protein 10 (IP-10) and cytokine responsive gene-2 (Crg-2).METHODS:The encoding genes of IP-10 and Crg-2 were amplified by RT-PCR from cultured human fibroblast cells and Balb/c mouse liver treated by IFN-gamma and TNF-alpha,respectively, and cloned into plasmids of pUC19 and pGEM3Zf(+).RESULTS:The nucleotide sequences of the amplified DNA were confirmed by endonucleases digestion and sequencing.CONCLUSION:Recombinant IP-10/crg-2 gene clones with 306bp and 314bp inserts were established for further research on biological activities and ligands of hIP-10/mCrg-2.

Journal Article↗
PubMed1998

Two novel gastric cancer-associated genes identified by differential display.

AIM:To clone novel gastric cancer-associated genes and investigate their roles in gastric cancer occurrence.METHODS:A method called differential display was used which allows the identification of differentially expressed genes by using PAGE to display PCR-amplified cDNA fragments between gastric cancer cells and normal gastric mucosa cells. These fragments were cloned into plasmid vector pUC18. Homology analysis was made after sequencing these fragments.RESULTS:Two novel genes were identified compared with sequences from GenBank. One was registered with the AD number AF 051783.In situ hybridization showed that these two novel genes expressed specifically in gastric cancer tissues.CONCLUSION:The two novel genes obtained by differential display were confirmed to be gastric cancer-associated genes using in situ hybridization.

Journal Article↗
PubMed1998

Immuno-protective effect of tumor cell vaccine on Kunming mice bearing Ehrlich ascites tumor.

AIM:To evaluate the immunity of chemically modified tumor cell vaccine.METHODS:Tumor cell vaccines (TCV) were prepared by incubating the live Ehrlich ascites tumor cells with concanavalin A-mitomycin C (ConA-MMC), mitomycin C (MMC), concanavalin A-glutaraldehyde (ConA-Glu), glutaraldehyde (Glu), or paraformaldehyde (Para), respectively. The whole cell or soluble forms of the vaccines were administered intraperitoneally into Kunming mice once a week for three times prior to the intraperitoneal inoculation of a lethal dose of live tumor cells. A second challenge with live tumor cells was given four weeks later. Survival and antibody production of the mice were analyzed.RESULTS:After the first challenge, the mice, received whole TCV of ConA-MMC, MMC (P < 0.01) and Glu (P < 0.05) promoted survival incidence than the controls. All the treated mice had the survival time prolonged. ConA-MMC vaccine treated mice had longer survival days than that of ConA-Glu ones (P < 0.05). For the soluble TCV immunized mice,those treated with vaccines of Para (P < 0.01), ConA-Para and ConA-Glu (P < 0.05) had longer survival periods compared with that of the controls. Following the second challenge, survival incidence of the mice received vaccines of ConA-MMC, MMC, ConA-Glu or Glu was significantly increased (P < 0.01). Moreover, all the treated mice had the survival time prolonged, and ConA-MMC vaccine treated mice had longer survival days than that of Para treated ones (P < 0.05). Antibodies against Ehrlich ascites tumor cells were found to be positive in sera of the mice treated with whole TCV of ConA-MMC.CONCLUSION:Ehrlich ascites tumor cells are immunogenic when treated with ConA-MMC, MMC, ConA-Glu, Glu or Para, which might act as safe and effective tumor vaccines with safety and effectiveness.

Journal Article↗
PubMed1998

Transduction of Fas gene or Bcl-2 antisense RNA sensitizes cultured drug resistant gastric cancer cells to chemotherapeutic drugs.

AIM:To compare the expression level of Fas gene and Bcl-2 gene in gastric cancer cells SGC7901 and gastric cancer MDR (multidrug resistant) cells SGC7901/VCR,to transduce Fas cDNA and Bcl-2 antisense nucleic acid into SGC7901/VCR cells respectively, and to observe the expression of two genes in transfectants and non-transfectants as well as their drug sensitivity.METHODS:Eukaryotic expression vector pBK-Fas cDNA and pDOR-anti Bcl-2 were constructed and transfected into SGC7901/VCR cells by lipofectamine,respectively.Northern blot and Western blot were used to detect the expression of mRNA and protein in SGC7901/VCR and SGC7901 cells and transfectants, and drug sensitivity of transfectants for VCR, CDDP and 5-FU was analyzed with MTT assay.RESULTS:After gene transfection, 80 for Fas and 120 for antisense Bcl-2 drug-resistant clones were selected from 2 X10(5) cells, transfection rate being 0.04% and 0.06%. Two clones of SGC7901 Fas/VCR cells and SGC7901 anti Bcl-2/VCR cells were randomly selected for further incubation. Hybridization results showed that the expression level of Fas mRNA and protein in SGC7901/VCR cells was much lower,but that of Bcl-2 mRNA and protein was higher than that in SGC7901 cells. The expression of Fas mRNA and protein in SGC7901 Fas/VCR cells was higher,and of Bcl-2 mRNA and protein was lower in SGC7901 anti Bcl-2/VCR cells than that in non-transfectants. MTT assay showed that transfectants were more sensitive to VCR, CDDP, 5-FU than non-transfectants. CONCLUSION:Bcl-2 gene displayed high expression while Fas gene had low expression in drug resistant gastric cancer cells. Expression of Bcl-2 protein was effectively blocked in SGC7901 anti Bcl-2/VCR cells by gene transfection. In contrast, the expression of Fas mRNA and protein in SGC7901 Fas/VCR cells increased. Fas gene and Bcl-2 antisense nucleic acid transfection sensitized drug resistant gastric cancer cells to chemotherapeutic drugs. These results suggest cell apoptosis plays an important role in the mechanism of MDR, and enhancing apoptosis might reverse MDR.

Journal Article↗