Inhibition of Benzo(a)pyrene-induced Lung Tumorigenesis in A/J Mice by Dietary N-Acetylcysteine Conjugates of Benzyl and Phenethyl Isothiocyanates during the Postinitiation Phase Is Associated with Activation of Mitogen-activated Protein Kinases and p53 Activity and Induction of Apoptosis

Increased level of MAP kinase phosphorylation in lung tissue of mice treated with ITC-NAC. Lung tissues were obtained on day 21 of the bioassay. Western blot analysis was performed with total proteins obtained from lung tissue of each treatment group. Antibodies used for A was anti-phospho-JNK1 and 2; B was anti-JNK 1; C was anti-phospho-p38 MAP kinase; and D was anti-phospho-Erk1 and 2. The first lane (3T3/UV) is the total protein isolated from NIH 3T3 cells 15 min after UV irradiation as a positive control.

Activation of p53 phosphorylation and increased expression of p21^WAF1/CIP1 and Bax in lung tissue from mice treated with ITC-NAC. Western blot analysis was performed with anti-phospho-p53 and anti-p53 antibodies using total proteins of lungs. A, phosphorylation on Ser-15. The upper blot was probed with anti-phospho-p53 (Ser-15) antibody, and the lower blot shows same membrane probed with anti-p53 antibody. Recombinant p53 serves as a positive control for p53 and a negative control for phospho-p53. B, phosphorylation levels of the serine residues 6, 9, 15, 20, and 392. The Y-axis is the fold of phosphorylation level over that of untreated control. The measurements were based on densitometry of phospho-p53 bands on Western blot. □, B(a)P; ▧, B(a)P + BITC-NAC; ▪, B(a)P + PEITC-NAC; ;, untreated. Western blot analysis was performed with anti-p21^WAF1/CIP1 (C) and anti-Bax (D) antibodies using total proteins of lungs. NS, a nonspecific band. UV-treated 3T3 cells in D serve as a positive control.

ITC-NAC treatment activates binding of AP-1 target sequence. EMSA was performed with AP-1 and NFκB target sequences using total proteins of lungs from each group. A-I and B-I demonstrate binding activity to AP-1 and NFκB, respectively. In the binding competition, assays used the total protein from the PEITC-NAC group (A-II) and the protein from untreated group (B-II). The protein in Lane Cold AP-1 or Lane Cold NFκB was preincubated with 10-fold amounts (4 ng) of unlabeled AP-1 (A-II) or NFκB (B-II) for 10 min before addition of ³²P-labeled probe. Proteins in Lane Non-specific DNA were preincubated with 10× nonspecific DNA sequences for 10 min. The last lane was obtained from the preincubation with mutant AP-1 (A-II) or mutant NFκB (B-II).