Transcriptional targeting of human liver carboxylesterase (hCE1m6) and simultaneous expression of anti-BCRP shRNA enhances sensitivity of breast cancer cells to CPT-11
| Autor: | Mukti N, Mishra, Mukthi N, Mishra, Kiran K, Vangara, Srinath, Palakurthi |
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| Rok vydání: | 2014 |
| Předmět: |
Reverse Transcriptase Polymerase Chain Reaction
Blotting Western Apoptosis Breast Neoplasms Flow Cytometry Irinotecan Real-Time Polymerase Chain Reaction Antineoplastic Agents Phytogenic Drug Resistance Multiple Neoplasm Proteins Gene Expression Regulation Neoplastic Drug Resistance Neoplasm Mutation Tumor Cells Cultured ATP Binding Cassette Transporter Subfamily G Member 2 Humans ATP-Binding Cassette Transporters Camptothecin Female RNA Messenger RNA Small Interfering Carboxylic Ester Hydrolases Cell Proliferation |
| Zdroj: | Anticancer research. 34(11) |
| ISSN: | 1791-7530 |
| Popis: | The major factor limiting the efficacy of breast cancer chemotherapy is multidrug resistance due to overexpression of the breast cancer resistance protein ATP-binding cassette, sub-family G (WHITE), member 2 (ABCG2). We hypothesized that conversion of camptothecin-11 (CPT-11) to its highly cytotoxic metabolite SN-38 by a mutant human carboxyl esterase (hCE1m6) specifically in cancer cells and inhibition of ABCG2 by anti-ABCG2 short hairpin RNA, leads to accumulation of a higher concentration of SN-38, resulting in higher therapeutic efficacy and less toxicity to normal cells.A mutant human carboxyl esterase hCE1m6 with human telomerase reverse transcriptase promoter was integrated into the VISA (VP16-Gal4-WPRE) amplification system. The plasmid was transfected into MCF-12A, MDA-MB-231, and MCF-7 cells using JetPRIME®. Cancer-specific expression of hCE1m6 in breast cancer cell lines was tested by real-time polymerase chain reaction (real time-PCR) and western blot. In vitro conversion of CPT-11 to SN-38 was evaluated on lysates of transfected cells. Cytotoxicity of CPT-11 against cells transfected with the plasmid was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Real-time PCR and western blot analysis revealed that hCE1m6 was expressed only in breast cancer cells, MCF-7 and MDA-MB-231, but not in the normal MCF-12A breast cell line. From the CPT-11 conversion assay on cell lysates, it was found that expressed hCE1m6 in cancer cells was able to effectively convert CPT-11 to SN-38.Breast cancer cell lines transfected with hCE1m6 showed an increased susceptibility to CPT-11 in comparison to MCF-12A cells. |
| Databáze: | OpenAIRE |
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