title: 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.
abstract: 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 beta (TGF-beta) signaling in POH-mediated carcinoma regression was explored. RNA expression studies, again based on a multiplexed-nuclease protection assay, showed that TGF-beta-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-beta1 was induced within 24 h of chemotherapy; (c) the mannose 6-phosphate/insulin-like growth factor II receptor and the TGF-beta 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-beta1, the mannose 6-phosphate/insulin-like growth factor II receptor, the TGF-beta type I and II receptors, and Smad2/Smad3 in epithelial cells. Nuclear localization of Smad2/Smad3 indicated that the TGF-beta 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.
authors.affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin-Medical School, Madison 53706, USA.
authors.forename: E A
authors.initials: EA
authors.lastname: Ariazi
authors.forename: Y
authors.initials: Y
authors.lastname: Satomi
authors.forename: M J
authors.initials: MJ
authors.lastname: Ellis
authors.forename: J D
authors.initials: JD
authors.lastname: Haag
authors.forename: W
authors.initials: W
authors.lastname: Shi
authors.forename: C A
authors.initials: CA
authors.lastname: Sattler
authors.forename: M N
authors.initials: MN
authors.lastname: Gould
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis; D002453:Cell Cycle / Q000187:drug effects; D005260:Female; D015674:Mammary Neoplasms, Animal / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D039821:Monoterpenes; D012334:RNA, Neoplasm / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects*; D013729:Terpenes / Q000494:pharmacology* / Q000627:therapeutic use; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D039821:Monoterpenes; D012334:RNA, Neoplasm; D013729:Terpenes; D016212:Transforming Growth Factor beta; C032208:perillyl alcohol