ABSTRACT
Our previous study demonstrated that BM-cyclin 1, a traditional anti-mycoplasma drug, could effectively reverse the multidrug resistance (MDR) of C-A120 cells. The present study aims to explore the reversal effect of BM-cyclin 1 on MDR and its mechanisms in BALB/C nude mice bearing C-A120 cells. Immunoblotting analysis and reverse transcription-polymerase chain reaction (RT-PCR) were used to study the change in multidrug resistance-associated protein 2 (MRP2) induced by BM-cyclin 1. We found that the expression levels of MRP2 protein and mRNA in C-A120 cells treated with BM-cyclin 1 were reduced significantly. Chemical colorimetry revealed no significant change in the level of glutathione (GSH). In the xenograft model, the inhibitory rate of C-A120 cells growth in BM-cyclin 1 plus adriamycin (ADM) group was 52%, which was significantly higher than in control group (P<0.01). The immunoblotting and RT-PCR results conclusively demonstrated that BM-cycin 1 could significantly reduce the expression of MRP2 in transplanted tumor. In conclusion, BM-cyclin 1 could effectively reverse the MDR of C-A120 cells in vivo by suppressing the expression of MRP2.
Subject(s)
Animals , Humans , Mice , Antiprotozoal Agents , Pharmacology , Cell Line, Tumor , Diterpenes , Pharmacology , Down-Regulation , Doxorubicin , Pharmacology , Drug Resistance, Multiple , Mice, Nude , Minocycline , Pharmacology , Multidrug Resistance-Associated Proteins , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , Xenograft Model Antitumor AssaysABSTRACT
<p><b>OBJECTIVE</b>To observe SHP-1 protein expression in breast cancer cell line MDA-MB-231 before and after SHP-1 gene transfer and its effect on the proliferation of MDA-MB-231 cells.</p><p><b>METHODS</b>The eukaryotic expression vector pEGFP-C3-SHP-1 was constructed and transfected into breast cancer cell line MDA-MB-231 via Lipofectamine 2000, and the positive clones were selected using G418. SHP-1 expression in MDA-MB-231 cells was detected with immunocytochemistry and Western blotting, and the cell growth curve was observed using MTT assay.</p><p><b>RESULTS</b>SHP-1 was highly expressed in transfected MDA-MB-231 cells, whose proliferation was significantly inhibited (P<0.05).</p><p><b>CONCLUSION</b>SHP-1 gene transfer into MDA-MB-231 cells results in inhibition of the cell proliferation.</p>
Subject(s)
Female , Humans , Breast Neoplasms , Genetics , Pathology , Cell Line, Tumor , Cell Proliferation , Gene Transfer Techniques , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To construct a retrovirus-mediated expression system carrying human SHP-1 gene to transfer SHP-1 gene in human breast cancer MDA-MB-231 cells.</p><p><b>METHODS</b>The full-length SHP-1 gene fragment was amplified by RT-PCR from the total RNA extracted from human breast cancer cell line MCF-7 over-expressing SHP-1 protein. The gene fragment was inserted into the vector pLNCX2 to construct the recombinant retroviral plasmid, which was transfected into the packaging cell PT67 via Lipofectamine2000. A cell line stably producing the virus was selected with G418. MDA-MB-231 cells was infected with the virus, and the expression of SHP-1 gene in the positive cell clone was detected with Western blotting.</p><p><b>RESULTS</b>A 1.8 kb cDNA fragment of SHP-1 gene was obtained from MCF-7 cells and successfully inserted into the pLNCX2. A stable cell clone PT67/SHP-1 and virus supernatant were obtained. Expression of SHP-1 protein was detected in the cells infected with the virus.</p><p><b>CONCLUSION</b>The recombinant retroviral vector carrying SHP-1 gene has been successfully constructed and MDA- MB-231/SHP-1 cell line expressing SHP-1 has been obtained to allow further functional study of SHP-1 in breast cancer.</p>