ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of gene Af116609 on gastric cancer multi-drug resistance (MDR) by introducing it into gastric cancer multi-drug resistant (MDR) cell line SGC7901/VCR.</p><p><b>METHODS</b>Gene Af116609 was cloned from SGC7901/VCR by RT-PCR and its differential expression between gastric cancer MDR cells and its parental cells was displayed by Northern blot. The gene was introduced to gastric cancer cells by transfection of recombinant eukaryotic expression vector by electroporation. MTT assay in vitro was applied to investigate its effect on multi-drug resistance phenotype of gastric cancer cells.</p><p><b>RESULTS</b>The full length CDS of gene Af116609, as long as 327 bp, was cloned from gastric cancer MDR cell line SGC7901/VCR and its sequence was coincident with the hypothetical gene Af116609 in GenBank. It was overexpressed in MDR cells than its parental cells at mRNA level. In the MTT assay in vitro, the drug sensitive cells transfected with sense eukaryotic expression vector showed upregulated targeted gene, with increased resistance to vincristine, 5-fliorouracil and arabinoside, and decreased resistance to adriamycin, but no influence on resistance to methotrexate. However, the drug resistant cells transfected with anti-sense eukaryotic expression vector, showed down regulated targeted gene, with less resistance to all the five anticancer drugs to different degrees.</p><p><b>CONCLUSION</b>Gene Af116609 is related to MDR phenotype of gastric cancer cells and may become a candidate molecular target to reverse the MDR of gastric cancer.</p>
Subject(s)
Humans , Antineoplastic Agents, Phytogenic , Pharmacology , Autoantigens , Genetics , Cell Line, Tumor , Drug Resistance, Multiple , Genetics , Drug Resistance, Neoplasm , Genetics , RNA, Small Cytoplasmic , Genetics , Ribonucleoproteins , Genetics , Stomach Neoplasms , Genetics , Pathology , Vascular Endothelial Growth Factor A , Vincristine , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To study the effect of alternative splicing form -MAD2beta of mitotic arrest deficient protein 2 (MAD2) on the formation of multidrug resistance in human gastric adenocarcinoma cell SGC7901.</p><p><b>METHODS</b>RNA was extracted from a multidrug resistance cell line SGC7901/ADR. The full-length MAD2beta cDNA was obtained by RT-PCR and cloned into the pUCm-T vector, and then recombined into the eukaryotic expression vector pcDNA3.1 in forward direction. Subsequently, pcDNA3.1/MAD2beta vectors were then transfected into SGC7901 cells by lipofectamine. Sensitivity to drug was detected by MTT assay. Cell cycle alteration and intracellular fluorescence intensity were determined by FACS.</p><p><b>RESULTS</b>A fragment of 0.53 Kb was obtained and confirmed by DNA sequencing which was a new alternative splicing form of MAD2 named as MAD2beta. pcDNA3.1/MAD2beta transfected SGC7901 cells (SGC7901/MAD2beta) were more resistant to ADR, VCR and MMC than the control cells (SGC7901/pcDNA3.1), and also ADR fluorescence intensity of SGC7901/MAD2beta cells was lower (P < 0.05) than that of SGC7901/pcDNA3.1 cells.</p><p><b>CONCLUSION</b>MAD2beta could increase the multidrug resistance of SGC7901 cell line.</p>
Subject(s)
Humans , Adenocarcinoma , Metabolism , Pathology , Alternative Splicing , Antibiotics, Antineoplastic , Pharmacology , Antineoplastic Agents, Phytogenic , Pharmacology , Calcium-Binding Proteins , Genetics , Cell Cycle Proteins , Cell Line, Tumor , DNA-Binding Proteins , Genetics , Doxorubicin , Pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Genetics , Mad2 Proteins , Mitomycin , Pharmacology , Repressor Proteins , Smad2 Protein , Stomach Neoplasms , Metabolism , Pathology , Trans-Activators , Genetics , Transfection , Vincristine , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the differential expression of RPL6/Taxreb107 between drug-resistant gastric cancer cell line SGC7901/ADR and gastric cancer cell line SGC7901 as well as its correlation with multiple-drug resistance (MDR) in gastric cancer cells.</p><p><b>METHODS</b>Total RNA was extracted from SGC7901 and SGC77901/ADR, with internal control RT-PCR, Northern blot, gene cloning and expression, construction of eukaryotic expression vector, gene transfection by electroporation. The accumulation and retention of ADR in transiently transfected cell was detected by flow cytometry.</p><p><b>RESULTS</b>The internal control RT-PCR and Northern blot showed high RPL6/Taxreb107 expression in SGC7901/ADR cell line. Sense and antisense eukaryonic expression vectors demonstrated by double enzyme digestion were successfully transfected into gastric cancer cell line SGC7901 and SGC7901/ADR respectively by electroporation. The accumulation and retention of ADR detected 48 hours after transfection showed that RPL6 gene had shown effect on drug resistance in gastric cancer cell.</p><p><b>CONCLUSION</b>The high expression of RPL6/Taxreb107 in drug resistant gastric cancer cell shows its correlation with multiple-drug resistance in gastric cancer.</p>