Selective inhibition of MCF-7piGST breast tumors using glutathione transferase-derived 2-methylene-cycloalkenones

Journal of Medicinal Chemistry

Volumn 48, Issue 21, 2005, Pages 6549-6552

  Joseph, Erin ^b   Ganem, Bruce ^c   Eiseman, Julie L ^b   Creighton, Donald J ^a  

^a UNIVERSITY OF MARYLAND BALTIMORE COUNTY   (United States)

^b UNIVERSITY OF PITTSBURGH CANCER INSTITUTE   (United States)

^c Department of Obstetrics Gynecology   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

2 CROTONYLOXYMETHYL 2 CYCLOALKENONE DERIVATIVE; ALKYLATING AGENT; ANTINEOPLASTIC AGENT; GLUTATHIONE TRANSFERASE; PRODRUG; UNCLASSIFIED DRUG;

ANTINEOPLASTIC ACTIVITY; ARTICLE; BREAST CANCER; CANCER INHIBITION; CANCER RESISTANCE; CATALYSIS; CELL STRAIN MCF 7; CONTROLLED STUDY; CORRELATION ANALYSIS; DRUG CYTOTOXICITY; DRUG POTENCY; ENZYME ACTIVITY; HUMAN; HUMAN CELL; IC 50; PROTEIN EXPRESSION; PROTEIN FUNCTION;

ANTINEOPLASTIC AGENTS, ALKYLATING; BREAST NEOPLASMS; CELL LINE, TUMOR; CYCLOHEXANONES; DRUG RESISTANCE, MULTIPLE; DRUG RESISTANCE, NEOPLASM; DRUG SCREENING ASSAYS, ANTITUMOR; FEMALE; GLUTATHIONE S-TRANSFERASE PI; HUMANS; PRODRUGS; STRUCTURE-ACTIVITY RELATIONSHIP;

EID: 27144514444     PISSN: 00222623     EISSN: None     Source Type: Journal    
DOI: 10.1021/jm058245f     Document Type: Article

References (22)

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22. Approximately 40 μg of protein was loaded into each lane containing cell lines. Lane 1 contains the earliest passage in culture of the MCF-7/7pi stably transfected cell line (culture passage 2). Since the parental cell line, MCF-7/wt, grows more rapidly in culture, more cells were available from culture passage 2 for this cell line and the lysate from this cell line is presented in Lanes 2 and 3. Lane 4 is a later culture passage, passage 9, for the MCF-7/7pi to ensure that GST-pi is still expressed. Lane 5 is passage 11 for the parental line, MCF-7/wt, harvested on the same day as MCF-7/7pi passage 9. Lane 6 is the murine cell line, B16 melanoma, which has high concentrations of murine GST-pi. Lane 7 contains L1210 murine leukemia cells, which have low concentrations of murine GST-pi. Lane 8 contains the purified human GST-pi, which is the positive control. The later culture passages of MCF-7/7pi and MCF-7/wt were included to confirm that the expression of the stably transfected GST-pi does not change with passage. The murine cell lines were included to check for cross-reactivity of the antibodies and to confirm the concentrations of murine GST-pi in these cell lines. GAPDH, glyceraldehyde-3-phosphate dehydrogenase, served as the loading control for all cell lines. GAPDH is constitutively expressed in almost all tissues at high levels. Therefore, it is a useful marker when a loading/positive control is required in western blotting. The activity of GST-pi in each cell line was expressed as the ratio of GST-pi to GAPDH. Antihuman glutathione S-transferease P1-1 was obtained from Oxford Biomedical Research (antibody GS 72, Oxford, MI) and used at a dilution of 1/1000. Anti-GAPDH was obtained from Chemicom International (MAB374, Temecula, CA). Western blot method: Cell pellets were lysed in 10 vol of lysis buffer (50 mM Tris-HCl (pH 7.9) 100 mM NaCl, 1% NP-40m 2mM EDTA, 100mM NaF) containing fresh protein inhibitors (1 μg/mL pepstatin, 10 μg/mL aprotin, 5 μg/mL leupeptin, 5 mM PMSF, 0.1 μM microcystin, and 5 mM Na pyrophosphate). The tissue lysate was centrifuged at 13000g for 10 min. The supernatant was collected and protein concentration determined using a Bio-Rad protein assay system (Bio-Rad, Hercules, CA). Concentrations of bovine serum albumin between 0.05 and 0.5 mg/mL were used to obtain the calibration standard curve. Equal amounts of protein (40 μg) were denatured in 3X modified Laemmli sample buffer (Bio-Rad) and loaded on 4-15% gradient gels (Bio-Rad). The separated proteins were transferred to PVDF membranes and blotted with 5% nonfat milk in TBS for 1 h. The membranes were incubated with antibodies against GST-pi or GAPDH overnight. The immunoreactive signals were detected by ELC detection reagents (PerkinElmer Life Sciences, Boston, MA) following the manufacturer's instructions. The densities of the signals were quantified by densitometry with UN-SCAN-IT (Silk Scientific, Orem, UT).