Purpose-The anti-cancer effect of carnosol was investigated in human prostate cancer PC3 cells.Methods-Biochemical analysis and protein array data of carnosol treated PC3 cells were analyzed.Results-We evaluated carnosol for its potential anti-cancer properties in the PC3 cells. Using an MTT assay we found that carnosol (10 -70 µM) decreases cell viability in a time and dose dependent manner. Next, we evaluated the effect of carnosol (20-60 uM) effect using flow cytometry as well as biochemical analysis and found induction of G2-phase cell cycle arrest. To establish a more precise mechanism, we performed a protein array that evaluated 638 proteins involved in cell signaling pathways. The protein array identified 5'-AMP-activated protein kinase (AMPK), a serine/threonine protein kinase involved in the regulation of cellular energy balance as a potential target. Further downstream effects consistent with cancer inhibition included the modulation of the mTOR/HSP70S6k/4E-BP1 pathway. Additionally, we found that carnosol targeted the PI3K/Akt pathway in a dose dependent manner.Conclusions-These results suggest that carnosol targets multiple signaling pathways that include the AMPK pathway. The ability of carnosol to inhibit prostate cancer in vitro suggests carnosol may be a novel agent for the management of PCa.

Emerging data are suggesting that estrogens, in addition to androgens, may also be contributing to the development of prostate cancer (PCa). In view of this notion, agents that target estrogens, in addition to androgens, may be a novel approach for PCa chemoprevention and treatment. Thus, the identification and development of nontoxic dietary agents capable of disrupting androgen receptor (AR) in addition to estrogen receptor (ER) could be extremely useful in the management of PCa. Through molecular modeling, we found that carnosol, a dietary diterpene, fits within the ligand-binding domain of both AR and ER-α. Using a time-resolved fluorescence resonance energy transfer assay, we found that carnosol interacts with both AR and ER-α and additional experiments confirmed that it functions as a receptor antagonist with no agonist effects. LNCaP, 22Rv1, and MCF7 cells treated with carnosol (20-40 μmol/L) showed decreased protein expression of AR and ER-α. Oral administration of carnosol at 30 mg/kg 5 days weekly for 28 days to 22Rv1 PCa xenografted mice suppressed tumor growth by 36% (P = 0.028) and was associated with a decrease in serum prostate-specific antigen by 26% (P = 0.0042). These properties make carnosol unique to any known antiandrogen or antiestrogen investigated thus far for the simultaneous disruption of AR and ER-α. We suggest that carnosol may be developed or chemically modified through more rigorous structure-activity relationship studies for a new class of investigational agents-a dual AR/ER modulator. Cancer Prev Res; 3(9); 1112-23. ©2010 AACR.

A44 Emerging evidence suggest that androgens may not be the only hormone responsible for the etiology of prostate cancer (PCa). Recent data has suggested that estrogens may be another important piece to the PCa puzzle. For example the use of the Selective Estrogen Receptor Modulator (SERM) toremifene in HG-PIN patients for one year resulted in a 21% reduction in the conversion of HG-PIN to PCa after one year of treatment. It is well established that race correlates with the levels of androgens with African Americans having the highest, followed by Caucasians and lowest in Japanese males. A similar trend has been observed in regard to serum estrogen levels and it is noteworthy that with age estrogen levels remain constant while androgens levels decrease. Given that experimental animal models suggest that both estrogens and androgens are necessary for PCa development it may be a reasonable approach to simultaneously disrupt both androgen and estrogen signaling in PCa. Several SERMs that include tamoxifen, toremifine, and fulvestrant have been evaluated for their ability to also target the AR and function as a dual AR and ER antagonist, however, experiments have suggested these effects are saturable and in some instances SERMs exert AR agonist properties. In our quest to find a dual AR and ER antagonist present in dietary substances that humans consume we found that the dietary diterpene Carnosol displays growth inhibitory properties in the hormone sensitive prostate (LNCaP and CWR22Rv1) and breast (MCF7 and AU565) cancer cell lines. Given the structural similarity between Carnosol to dihydrotestosterone and estradiol we hypothesized that it may interact with both the androgen and estrogen receptor alpha (ER-α). Using a TR-FRET cell free biochemical assay we found that Carnosol can interact with both AR and ER-alpha with similar potencies. Next, we evaluated the antagonist and agonist properties of Carnosol using a functional assay where the AR and ER-alpha ligand binding domains are expressed as a fusion protein in HEK293 cells. We observed that Carnosol exhibits antagonist properties that are dose responsive and even more interestingly, regardless of dose, did not exert agonist properties. In LNCaP, CWR22Rv1, and MCF7 cells Carnosol was found to disrupt both androgen and estrogen signaling in a dose and time dependent manner. Our finding that Carnosol displays the ability to disrupt both androgen and estrogen signaling simultaneously suggesting that Carnosol may be developed or chemically modified through more rigorous structure activity relationship studies of other such chemical entities for a new class of investigational agents - a dual AR/ER modulator. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A44.