Activation of the Transforming Growth Factor β Signaling Pathway and Induction of Cytostasis and Apoptosis in Mammary Carcinomas Treated with the Anticancer Agent Perillyl Alcohol

The mechanisms of action of the anticancer agent perillyl alcohol (POH), presently in Phase II clinical trials, were investigated in advanced rat mammary carcinomas. Gross and ultrastructural morphology of POH-mediated tumor regression indicated that apoptosis accounted for the marked reduction in the epithelial compartment. Characterization of cell growth and death indices revealed that apoptosis was induced within 48 h of chemotherapy, before the induction of cytostasis. RNA expression studies, based on a multiplexed-nuclease protection assay, demonstrated that cell cycle- and apoptosis-related genes were differentially expressed within 48 h of POH treatment; p21 Cip1/WAF1 , bax , bad , and annexin I were induced; cyclin E and cyclin-dependent kinase 2 were repressed; and bcl -2 and p53 were unchanged. Next, a potential role for transforming growth factor β (TGF-β) signaling in POH-mediated carcinoma regression was explored. RNA expression studies, again based on a multiplexed-nuclease protection assay, showed that TGF-β-related genes were induced and temporally regulated during POH treatment: ( a ) c-jun and c-fos were transiently induced within 12 h of chemotherapy; ( b ) TGF-β1 was induced within 24 h of chemotherapy; ( c ) the mannose 6-phosphate/insulin-like growth factor II receptor and the TGF-β type I and II receptors were induced within 48 h of chemotherapy; and ( d ) smad3 was induced during active carcinoma regression. In situ protein expression studies, based on fluorescence-immunohistochemistry in concert with confocal microscopy, confirmed up-regulation and demonstrated colocalization of TGF-β1, the mannose 6-phosphate/insulin-like growth factor II receptor, the TGF-β type I and II receptors, and Smad2/Smad3 in epithelial cells. Nuclear localization of Smad2/Smad3 indicated that the TGF-β signaling pathway was activated in regressing carcinomas. Subpopulations of Smad2/Smad3-positive and apoptotic nuclei colocalized, indicating a role for Smads in apoptosis. Thus, Smads may serve as a potential biomarker for anticancer activity. Importantly, none of the POH-mediated anticancer activities were observed in normal mammary gland.