title: Transcriptional targeting of human liver carboxylesterase (hCE1m6) and simultaneous expression of anti-BCRP shRNA enhances sensitivity of breast cancer cells to CPT-11.
abstract: 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.
  
  
  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 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.
  
  
  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.
  
  
  CONCLUSIONS
  Breast cancer cell lines transfected with hCE1m6 showed an increased susceptibility to CPT-11 in comparison to MCF-12A cells.
authors.affiliation: Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, TX, U.S.A.
authors.forename: Mukti N
authors.initials: MN
authors.lastname: Mishra
authors.forename: Mukthi N
authors.initials: MN
authors.lastname: Mishra
authors.affiliation: Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, TX, U.S.A.
authors.forename: Kiran K
authors.initials: KK
authors.lastname: Vangara
authors.affiliation: Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, TX, U.S.A. palakurthi@tamhsc.edu.
authors.forename: Srinath
authors.initials: S
authors.lastname: Palakurthi
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D000077146:Irinotecan; D009154:Mutation / Q000235:genetics*; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000972:Antineoplastic Agents, Phytogenic; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000077146:Irinotecan; D002265:Carboxylic Ester Hydrolases; C474609:CES1 protein, human; D002166:Camptothecin
keywords: BCRP-shRNA; CPT-11; Transcriptional targeting; breast cancer cells; drug resistance; human carboxylesterase; transfection