title: 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.
abstract: 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.
authors.affiliation: Department of Molecular Physiology and Biological Physics and Cancer Center, University of Virginia, Health Sciences Center, Charlottesville, VA 22908, USA.
authors.forename: Dora
authors.initials: D
authors.lastname: Bigler
authors.forename: Kay M
authors.initials: KM
authors.lastname: Gulding
authors.forename: Roger
authors.initials: R
authors.lastname: Dann
authors.forename: Fayad Z
authors.initials: FZ
authors.lastname: Sheabar
authors.forename: Mark R
authors.initials: MR
authors.lastname: Conaway
authors.forename: Dan
authors.initials: D
authors.lastname: Theodorescu
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology*; D000726:Androgen Antagonists / Q000494:pharmacology; D000728:Androgens; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology* / Q000592:standards; D002851:Chromatography, High Pressure Liquid; D004054:Diethylstilbestrol / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D004365:Drugs, Chinese Herbal; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D009376:Neoplasms, Hormone-Dependent / Q000235:genetics / Q000473:pathology*; D010936:Plant Extracts / Q000494:pharmacology* / Q000592:standards; D011401:Promoter Regions, Genetic / Q000187:drug effects* / Q000235:genetics; D017430:Prostate-Specific Antigen / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000235:genetics / Q000473:pathology*; D012045:Regulatory Sequences, Nucleic Acid / Q000187:drug effects
chemical_list: D000726:Androgen Antagonists; D000728:Androgens; D018931:Antineoplastic Agents, Hormonal; D000972:Antineoplastic Agents, Phytogenic; D004365:Drugs, Chinese Herbal; D009363:Neoplasm Proteins; D010936:Plant Extracts; C472637:herbal preparation PC-CARE; C114209:herbal preparation PC-SPES; D004054:Diethylstilbestrol; D008156:Luciferases; D017430:Prostate-Specific Antigen