Gene profiling and promoter reporter assays: novel tools for comparing the biological effects of botanical extracts on human prostate cancer cells and understanding their mechanisms of action.

The use of botanical mixtures is commonplace in patients with prostate cancer, yet the majority of these products have not been rigorously tested in clinical trials. Here we use PC-SPES, a combination of eight herbs that has been shown to be effective in clinical trials in patients with prostate cancer, as a model system to demonstrate 'proof of principle' as to how gene expression profiling coupled with promoter assays can evaluate the effect of herbal cocktails on human prostate cancer. In addition, we demonstrate how such approaches may be used for standardization of herbal extract activity by comparing the gene profile of PC-SPES with that of PC-CARE, a product with a similar herbal composition. Since prior studies have shown that PC-SPES contains estrogenic organic compounds, and such compounds are known to affect prostate cancer, an important issue is whether these are the primary drivers of the gene profile. Our data suggest that gene expression profiles of LNCaP human prostate cancer cells in response to PC-SPES are different from those found when diethylstilbestrol (DES), a synthetic estrogen, is used, suggesting that the estrogenic moieties within PC-SPES do not drive this expression signature. In contrast, the expression profile of PC-CARE was almost identical to that of DES, highlighting that mixtures containing similar herbal compositions do not necessarily result in similar biological activities. Interestingly, these three agents cause similar in vitro morphological changes and growth effects on LNCaP. To validate the expression profiling data, we evaluated the protein expression and promoter activity of prostate-specific antigen (PSA), a gene induced by PC-SPES but repressed by DES. In order to gain a mechanistic understanding of how PC-SPES and DES affect PSA expression differently, LNCaP cells were transiently transfected with wild-type and mutagenized PSA promoter, ARE concatemers and appropriate controls. We provide evidence that androgen response elements (ARE) II and III within the promoter region are responsible for the suppressive effects of DES and stimulatory effects of PC-SPES. In addition, we show that the effects on PSA transcription are ARE specific in the case of DES while PC-SPES affects this promoter nonspecifically. In conclusion, expression profiling coupled with mechanistic target validation yield valuable clues as to the mode of action of complex botanical mixtures and provides a new way to compare objectively mixtures with similar components either for effect or quality assurance prior to their use in clinical trials.

The role of Ral A in epidermal growth factor receptor-regulated cell motility.

Tumor cell motility is one of the rate-limiting steps of invasion, which defines progression toward a more malignant phenotype. Elevated expression of epidermal growth factor (EGF) receptor in many cancers is associated with progression of superficial to invasive forms of the disease and is sometimes found in tumors that also have activating Ras mutations, suggesting that both events contribute to tumor invasion. Here we show that EGF stimulates motility in human tumor cell lines, which harbor activating Ha-RasV12 via a novel signal transduction pathway mediated by the small GTP-binding proteins RalA and RhoA but independent of Rac1 and Cdc42. On EGF stimulation, RalA localizes to the cell membrane. In addition, activation of RalA and expression of Rho were increased by EGF stimulation in both the nonmetastatic and metastatic variants of the same cell line. However, elevated levels of constitutively activated RalA were only found in the metastatic variant. This is the first demonstration of an essential role for Ral in EGF-mediated cell motility and its potential contribution to tumor metastasis in human cancer.