Search

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.

Monoterpenes, including limonene and its in vivo rat plasma metabolites, have been shown to be inhibitors of protein isoprenylation of small G proteins, including p21 ras. In addition, dietary limonene has been shown to be capable of preventing the development and causing the regression of chemically induced mammary carcinomas, many of which contain activated ras oncogenes. On the basis of these observations, it was hypothesized that a possible mechanism by which limonene exerts its effects on the chemoprevention and regression of mammary tumors involves the inhibition of protein isoprenylation of the small G protein p21. In the first study, we asked whether dietary limonene was able to prevent the development of mammary carcinomas which were induced using direct retroviral gene transfer of v-Ha-ras into the mammary parenchyma in situ. Limonene modified neither the rate of gene transfer nor the stability of gene expression. However, limonene did greatly inhibit the formation of mammary carcinomas induced by the insertion of activated ras. In a follow-up study, we asked whether chemoprevention by limonene was preferentially effective against a subset of chemically induced mammary carcinomas with activated ras. Rats were fed limonene to prevent the development of N-nitroso-N-methylurea-induced mammary tumors, a majority of which contain the activated Ha-ras oncogene. As expected, limonene administration increased the latency period and lowered the frequency of mammary carcinoma development as compared to controls. However, tumor characterization revealed that limonene treatment did not alter the percentage of carcinomas with activated ras. These studies are consistent with the above studies in that limonene is effective in preventing mammary carcinomas with activated ras. Interestingly, carcinomas without activated ras were prevented to the same extent as those with the activated oncogene.

The monoterpenes represent a potentially new class of breast cancer therapeutic agents. We have shown that d-limonene induces the regression of advanced rat mammary adenocarcinomas. These regressing tumors have an increased cellular concentration of both the mannose-6-phosphate/insulin-like growth factor II receptors and transforming growth factor beta 1. The terpene-induced regression of mammary tumors may result in part from the mitoinhibitory and differentiation properties of active transforming growth factor beta 1. Furthermore, the activation of transforming growth factor beta 1 in these tumors is likely to be facilitated by the increased concentration of the mannose-6-phosphate/insulin-like growth factor II receptors in the mammary tumor cells. Tumors not responding to terpene therapy lacked a rise in the mannose-6-phosphate/insulin-like growth factor II receptor level which may relate to the fact that this gene is hemizygous due to maternal imprinting.

Dietary administration of the monocyclic monoterpenoid d-limonene causes complete regression of both dimethylbenz[alpha]anthracene- and N-nitroso-N-methylurea-induced rat mammary carcinomas. Carcinomas regress when limonene is added to the diet either when the tumor is small and still capable of spontaneously regressing or when it is large and progressed beyond the stage when it is susceptible to spontaneous regression. The limonene dose-tumor regression response relationship is steep. Significant regressions are not observed at 5% dietary levels, while a majority of tumors completely regress above a 7.5% dietary level. Limonene appears to act in a cytostatic fashion. Its removal from the diet results in a significant number of tumor recurrences. Regressing tumors have a unique histopathological appearance that is not associated with gross cytotoxicity, immune cell involvement, or apoptosis. Preliminary analysis suggests a remodeling/redifferentiation event underlying regression. The underlying mechanism of action of limonene in causing tumor regression is unknown. However, it should be noted that limonene can selectively inhibit the isoprenylation of small G proteins. Monoterpenoids such as limonene represent a novel class of anticancer drugs with the potential to cause tumor regressions with limited toxicity.