Transcriptional targeting of human liver carboxylesterase (hCE1m6) and simultaneous expression of anti-BCRP shRNA enhances sensitivity of breast cancer cells to CPT-11.

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 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. CONCLUSION: Breast cancer cell lines transfected with hCE1m6 showed an increased susceptibility to CPT-11 in comparison to MCF-12A cells.

Comparative evaluation of small-molecule chemosensitizers in reversal of cisplatin resistance in ovarian cancer cells.

Cisplatin-resistance is one of the major challenges in the treatment of epithelial ovarian cancer. Small-molecule chemosensitizers provide a therapeutically feasible approach to overcome cisplatin resistance in ovarian cancer. However, proper selection of chemosensitizer is of prime importance owing to phenotypic differences in cisplatin-resistant ovarian cancers. The resistance reversal activity of chemosensitizers buthionine sulfoximine (BSO), triethylenetetramine (TETA), genistein, rapamycin and colchicine was investigated in various cisplatin-resistant ovarian cancer cells, 2008 C13, CP70 and OVCAR 8 using MTT assays. Cellular accumulation of cisplatin in the presence of chemosensitizers was analyzed by inductively-coupled plasma-mass spectroscopy (ICP-MS). Chemosensitizers exhibited resistance reversal activity in 2008 C13 and CP70 cells in the following order; colchicine> genistein>TETA> rapamycin ≥ BSO (p<0.05), which is in correlation with cellular accumulation of cisplatin. In conclusion, our study demonstrates that resistance reversal activity of chemosensitizers varies with phenotypic behavior of cisplatin-resistant ovarian cancer cells. Data from our study can be utilized to choose a specific chemosensitizer for individualized combination therapy for cisplatin-resistant ovarian cancer.

Mechanism of gene transfection by polyamidoamine (PAMAM) dendrimers modified with ornithine residues.

The aim of this study was to prepare and investigate the mechanism of uptake of the dendriplexes prepared with ornithine-conjugated polyamidoamine (PAMAM) G4 dendrimers. Ornithine-conjugated PAMAMG4 dendrimers were prepared by Fmoc synthesis. A comparative transfection study in NCI H157G cells and polyamine transport-deficient cell line NCI H157R was performed to confirm the role of the polyamine transporter system (PAT) in the dendriplex uptake. Transfection efficiency significantly increased with increase in generation number and extent of ornithine conjugation. Transfection efficiency of the PAMAMG4-ORN60 dendrimers significantly decreased in presence of excess of ornithine (P < 0.05) and paraquat (P < 0.01) but not of PAMAMG4 dendrimers. Transfection efficiency of PAMAMG4-ORN60 was significantly low in NCI H157R (31.66 ± 3.95%, RFU: 17.87 ± 1.34) as compared to NCI H157G cell line (63.07 ± 6.8%, relative fluorescence units (RFU): 23.28 ± 0.66). Results indicate the role of PAT in addition to charge-mediated endocytosis in the internalization of ornithine-conjugated PAMAMG4 dendrimers. Cytotoxicity analysis (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay) in human embryonic kidney cell line (HEK) 293T cells showed that the dendriplexes were non-toxic at N/P 10.

Biotinylated PAMAM dendrimers for intracellular delivery of cisplatin to ovarian cancer: role of SMVT.

AIM: The aim of this study was to prepare biotinylated PAMAM dendrimers loaded with cisplatin and to evaluate the cytotoxicity in ovarian cancer cell lines. MATERIALS AND METHODS: Biotinylated and unconjugated dendrimer-cisplatin complexes were investigated for encapsulation efficiency, in vitro cytotoxic activity and cellular accumulation of cisplatin in OVCAR-3, SKOV-3, A2780 (wild-type) and CP70 (A2780/CP70, cisplatin-resistant) cells. RESULTS: Encapsulation efficiency of cisplatin ranged from 5.33% to 21.10%. In vitro cytotoxic activity revealed that IC(50) values of dendrimer-cisplatin complexes were significantly lower than that of free cisplatin in OVCAR-3, SKOV-3 and CP70 cell lines. Cellular uptake data showed highest accumulation of platinum by PAMAMG(4) NH(2) dendrimer complexes of cisplatin in A2780 (19.41±0.85 µg/ml) and CP70 (25.25±1.25 µg/ml) cell lines in comparison with cisplatin uptake of only 1.77±0.351 µg/ml in A2780 and 2.31±0.421 µg/ml in CP70 cells. CONCLUSION: In conclusion, biotinylated PAMAM dendrimers may be utilized as potential targeting agents for cisplatin delivery to ovarian cancer.