Anti-proliferative and pro-apoptotic effects of rosemary and constituent terpenoids in a model for the HER-2-enriched molecular subtype of clinical breast cancer

Neoadjuvant treatment options for human epidermal growth factor receptor-2 (HER-2)-enriched and luminal B molecular subtypes of clinical breast cancer include HER-2-targeted therapy with chemotherapy or anti-hormonal therapy. These treatment options result in systemic toxicity and acquired tumor resistance. Minimally toxic naturally occurring phytochemicals may represent testable alternatives to conventional therapy. HER-2-overexpressing tumorigenic human mammary epithelial 184-B5/HER cells represent a model for the HER-2-enriched breast cancer subtype. Non-fractionated rosemary extract (RME) and constituent phenolic terpenoids ursolic acid (UA), carnosol (CSOL) and carnosic acid (CA) represented the test agents. Anchorage-independent (AI) proliferation, cell cycle progression, cellular apoptosis and expression of cell cycle-regulatory and apoptosis-specific proteins represented the mechanistic end point biomarkers. Relative to the parental non-tumorigenic 184-B5 cells, tumorigenic 184-B5/HER cells exhibited decreased population doubling, increased saturation density, accelerated cell cycle progression and downregulated cellular apoptosis, confirming the loss of homeostatic control of proliferation. Treatment with the test agents resulted in a dose-dependent decrease in AI colony number, indicating a decrease in cancer risk. Mechanistically, RME and UA inhibited G(1)-S phase transition resulting in an increased G(1):S+G(2)/M ratio and decreased cyclin D1 expression. The pro-apoptotic effect of RME and UA was indicated by increased sub-G(0) (apoptotic) cell population, and relevant reciprocal modulation, as demonstrated by decreased anti-apoptotic B-cell lymphoma-2 (Bcl-2) and increased pro-apoptotic Bcl-2-associated X protein expression. In contrast, treatment with CA and CSOL resulted in cytostatic G(2)/M arrest and an increase in cyclin B1 expression; thus, naturally-occurring rosemary and its constitutive terpenoids re-establish homeostatic control of proliferation and decrease cancer risk via distinct mechanisms. These data validate an experimental approach to prioritize efficacious natural compounds as testable alternatives for conventional chemo-endocrine and HER-2-targeted therapies in HER-2-enriched breast cancer.