Identification of differentially expressed genes and activation of the TGF-beta signal transduction pathway in mammary carcinomas treated with the anticancer monoterpenes limonene and perillyl alcohol.

摘要

The monoterpenes limonene (LIM) and perillyl alcohol (POH) are effective chemoprevantitive and chemotherapeutic agents currently in clinical trials.;Using a newly developed methodology, subtractive display, numerous differentially expressed genes were identified in dietary 10% LIM-treated, DMBA-induced rat mammary carcinomas undergoing regression compared to control carcinomas. The subtractive display methodology combined a polymerase chain reaction-based subtractive hybridization procedure with the ability to selectively amplify and display subpopulations of subtracted cDNA for their subsequent identification. Involvement of the transforming growth factor beta (TGF-beta) signaling pathway in tumor regression was suggested by the identification of mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) and TGF-beta type II receptor (TbetaIIR) as induced genes. Involvement of apoptosis in tumor regression was suggested by identification of the mammary gland marker of programmed cell death, lipocortin 1, also termed annexin I, as an induced gene. Consistent with differentiation in tumor regression, YWK-II was induced and neuroligin 1 was repressed.;TGF-beta signaling, and its potential downstream effects on cellular proliferation and death, were investigated in 2% POH-treated regressing mammary carcinomas. Using nuclease protection assays (NPAs) to study RNA expression, it was found that TGF-beta signaling components were induced and temporally regulated: first, transient c-jun and c-fos; second, TGF-beta1; third, M6P/IGF2R, TbetaIIR, and TGF-beta type I receptor (TbetaIR); and fourth, smad3. Immunohistochemistry confirmed that TGF-beta1, M6P/IGF2R, TbetaIIR, TbetaIR, and Smad2/Smad3 were upregulated and colocalized in epithelium. Smad2/Smad3 exhibited nuclear localization, indicating activation of TGF-beta signaling in POH-treated carcinomas. A subpopulation of Smad2/Smad3 nuclei colocalized with a subpopulation of apoptotic nuclei. NPAs demonstrated that the cell cycle- and apoptosis-related genes p21Cip1/WAF1, bax, bad, and annexin I were induced, cyclin E and CDK2 were repressed; and bcl-2 and p53 were unchanged. As demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and in vivo bromodeoxyuridine labeling, POH induced apoptosis within 48 h of treatment, before the induction of cytostasis during active carcinoma regression. Importantly, all of POH's anticancer activities were specific to mammary carcinomas compared to normal mammary gland and thus may explain in part the lack of toxicity associated with monoterpene therapy and its high therapeutic efficacy in vivo.