TY - JOUR T1 - Transcriptional Targeting of Human Liver Carboxylesterase (hCE1m6) and Simultaneous Expression of Anti-BCRP shRNA Enhances Sensitivity of Breast Cancer Cells to CPT-11 JF - Anticancer Research JO - Anticancer Res SP - 6345 LP - 6351 VL - 34 IS - 11 AU - MUKTHI N. MISHRA AU - KIRAN K. VANGARA AU - SRINATH PALAKURTHI Y1 - 2014/11/01 UR - http://ar.iiarjournals.org/content/34/11/6345.abstract N2 - Background: 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. Materials and Methods: 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 JetPRIMEĀ®. 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. Results: 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. Conclusion: Breast cancer cell lines transfected with hCE1m6 showed an increased susceptibility to CPT-11 in comparison to MCF-12A cells. ER -