Activation of the transforming growth factor beta 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), pr esently in Phase II clinical trials, were investigated in advanced rat mamm ary carcinomas. Gross and ultrastructural morphology of POH-mediated tumor regression indicated that apoptosis accounted for the marked reduction in t he epithelial compartment. Characterization of cell growth and death indice s revealed that apoptosis was induced within 48 h of chemotherapy, before t he induction of cytostasis. RNA expression studies, based on a multiplexed- nuclease protection assay, demonstrated that cell cycle- and apoptosis-rela ted genes were differentially expressed within 48 h of POH treatment; p21(C ip1/WAF1), bar, bad, and annexin I were induced; cyclin E and cyclin-depend ent kinase 2 were repressed; and bcl-2 and p53 were unchanged. Next, a pote ntial role for transforming growth factor beta (TGF-beta) signaling in POH- mediated carcinoma regression was explored. RNA expression studies, again b ased on a multiplexed-nuclease protection assay, showed that TGF-beta-relat ed 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 -beta 1 was induced within 24 h of chemotherapy; (c) the mannose 6-phosphat e/insulin-like growth factor II receptor and the TGF-beta type I and II rec eptors were induced within 48 h of chemotherapy; and (d) smad3 was induced during active carcinoma regression, bt situ protein expression studies, bas ed on fluorescence-immunohistochemistry in concert with confocal microscopy , confirmed up-regulation and demonstrated colocalization of TGF-beta 1, th e mannose 6-phosphate/insulin-like growth factor II receptor, the TGF-beta type I and II receptors, and Smad2/Smad3 in epithelial cells. Nuclear Local ization of Smad2/Smad3 indicated that the TGF-beta signaling pathway was ac tivated in regressing carcinomas. Subpopulations of Smad2/Smad3-positive an d apoptotic nuclei colocalized, indicating a role for Smads in apoptosis. T hus, Smads may serve as a potential biomarker for anticancer activity. Impo rtantly, none of the POH-mediated anticancer activities were observed in no rmal mammary gland.