Chinese hamster V79 cell lines were constructed for stable expression of human cytochrome P450 1B1 (P450 1B1) in order to study its role in the metabolic activation of chemicals and toxicological consequences. The new V79 cell lines were applied to studies on DNA adduct formation of the polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P). This compound has been found to be an environmental pollutant, and in rodent bioassays it is the most carcinogenic PAH yet discovered. Activation of DB[a,l]P in various metabolizing systems occurs via fjord region DB[a,l]P-11, 12-dihydrodiol 13,14-epoxides (DB[a,l]PDE): we found that DB[a,l]P is stereoselectively metabolized in human mammary carcinoma MCF-7 cells to the (-)-anti- and (+)-syn-DB[a,l]PDE which both bind extensively to cellular DNA. To follow up this study and to relate specific DNA adducts to activation by individual P450 isoforms, the newly established V79 cells stably expressing human P450 1B1 were compared with those expressing human P450 1A1. DNA adduct formation in both V79 cell lines differed distinctively after incubation with DB[a,l]P or its enantiomeric 11,12-dihydrodiols. Human P450 1A1 catalyzed the formation of DB[a,l]PDE-DNA adducts as well as several highly polar DNA adducts as yet unidentified. The proportion of these highly polar adducts to DB[a,l]PDE adducts was dependent upon both the concentration of DB[a,l]P and the time of exposure. In contrast, V79 cells stably expressing human P450 1B1 generated exclusively DB[a,l]PDE-DNA adducts. Differences in the total level of DNA binding were also observed. Exposure to 0.1 microM DB[a,l]P for 6 h caused a significantly higher level of DNA adducts in V79 cells stably expressing human P450 1B1 (370 pmol/mg of DNA) compared to those with human P450 1A1 (35 pmol/mg of DNA). A 4-fold higher extent of DNA binding was catalyzed by human P450 1B1 (506 pmol/mg of DNA) compared to human P450 1A1 (130 pmol/mg of DNA) 6 h after treatment with 0.05 microM (-)-(11R,12R)-dihydrodiol. In cells stably expressing human P450 1B1 the DNA adducts were derived exclusively from the (-)-anti-DB[a,l]PDE. These results indicate that human P450 1B1 and P450 1A1 differ in their regio- and stereochemical selectivity of activation of DB[a,l]P with P450 1B1 forming a higher proportion of the highly carcinogenic (-)-anti-(11R, 12S,13S,14R)-DB[a,l]PDE metabolite.

Pancreatic cancer is one of the most aggressive human cancers with a 5-year survival rate of <5%. Overexpression of transforming growth factor-beta 2 (TGF-b2) in pancreatic malignancies is suggested to be a pivotal factor for malignant progression by inducing immunosuppression, metastasis, angiogenesis and proliferation. Trabedersen (AP 12009) is a phosphorothioate antisense oligodeoxynucleotide specific for human TGF-b2 mRNA and was successfully tested in a randomized, active-controlled phase IIb clinical study in patients with high-grade glioma. Here, we report on the antitumor activity of trabedersen in human pancreatic cancer cells and in an orthotopic xenograft mouse model of human metastatic pancreatic cancer. Trabedersen reduced TGF-b2 secretion in human pancreatic cell lines with an IC 50 in the low lM range without transfection reagent, clearly inhibited cell proliferation, and completely blocked migration of pancreatic cancer cells. Additionally, trabedersen reversed TGF-b2-mediated immunosuppression of pancreatic cancer cells targeted by lymphokine activated killer (LAK) cells, resulting in considerably increased LAK cell-mediated cytotoxicity. Moreover, in an orthotopic mouse model of metastatic pancreatic cancer, intraperitoneal (i.p.) treatment with trabedersen significantly reduced tumor growth, lymph node metastasis and angiogenesis. These promising results warrant further clinical development of trabedersen. (Cancer Sci 2011; 102: 1193-1200

A set of small nonpeptidic diaryl phosphonate inhibitors was prepared. Some of these inhibitors show potent and highly selective irreversible uPA inhibition. The biochemical and modeling data prove that the combination of a benzylguanidine moiety with a diaryl phosphonate ester results in optimized molecules for derivatizing the serine alcohol in the uPA active site. Selected compounds show significant antimetastatic effects in the BN-472 rat mammary carcinoma model. We report in this paper a preclinical proof of concept that selective, irreversible uPA inhibitors could be valuable in antimetastatic therapy.