The present study demonstrates the power of chemical-genetic profiling in yeast as a model system to predict potential human molecular targets of bioactive botanical products. First, using SJW, we show that the use of a high-density yeast barcode microarray has considerable power to identify SJW-sensitive yeast deletion strains not previously described as sensitive to SJW. Second, using a microarray containing multiple replicate barcode features, we show that the strains can be ranked according to their sensitivity to SJW. Finally, using computational comparative genomics techniques and human cell-based immunocytochemical assays, we demonstrate that the identification of SJW-sensitive genes in yeast with the barcode microarray facilitated the identification of SJW-sensitive orthologous human genes linked to angiogenesis, a process involved in wound recovery and tumor growth, and genes linked to the predisposition of neurodegenerative and psychiatric genetic diseases.
Yeast has long been used as a human genetic model system. It is easy to culture, genetically tractable, and has a genome with approximately 44% homology to the human genome. Comparative genomics techniques that can integrate and interrogate yeast and human molecular information can be used to elucidate the answers to functional genomics questions. Therefore, identification of botanical molecular targets in yeast may help identify potential orthologous targets in humans, based on the conservation of homologous genes and proteins throughout phylogeny. Interestingly, although sequence and structural aspects may be conserved throughout evolution, that is not always the case for molecular function, such that orthologs of molecular targets which mediate a protective function in yeast may have a dramatically different function in human physiology.
We identified 78 genes in yeast represented by SJW-sensitive strains for which the signal intensity in the untreated sample was higher than that for the treated sample (). Biological processes represented by genes in this list include transport, vesicle-mediated transport, signal transduction, protein modification, lipid metabolism, transcription, and translation, among others. In yeast, these genes are identified as serving an important protective role or essential function in the adaptive response(s) to the cytotoxic challenge incurred by SJW exposure.
Using sequence-based comparison techniques, we determined that 52 of the 78 SJW-sensitive yeast genes have human orthologs (). The top molecular functions were associated with cellular growth, development, assembly, and organization and cell death of brain and central nervous system cells. It should be noted that the yeast genome does not contain genes for cytochrome P450 enzymes, which may explain the absence of such genes from the list of human orthologs.
Four of the 52 human orthologs are associated with angiogenesis, the formation of new blood vessels. These orthologs include UBE2C, KRAS, MEK2, and EIF2B3. Computational analysis associated UBE2C with angiogenesis at the level of HIF1α degradation. The potential for HIF1α modulation by SJW was investigated by Western blot analysis of SJW-treated MDA-MB-231 cells. Protein expression of HIF1α increased after 19 h of exposure to SJW (). Computational analysis associated KRAS, MEK2, and EIF2B3 with angiogenesis at the level of VEGF signaling. The potential for VEGF modulation by SJW was investigated by Western blot analysis of the culture medium of MDA-MB-231 cells treated with SJW. VEGF protein levels in the culture medium of MDA-MB-231 cells increased after 19 h of treatment with SJW (). The ability of SJW to activate HIF1α and VEGF protein expression suggests a potential for SJW to promote wound healing, recovery, or cell growth processes through the activation of angiogenesis to increase blood flow through new blood vessel formation.
Six of the 52 identified human orthologs were associated with neurological diseases or psychiatric disorders. These genes include GIT1, HIRA, ACAA1, MPV17, HSPA5
, and SIRT2
. Our focus was attracted to SIRT2
, which encodes an NAD (+)-dependent protein deacetylase involved in alpha-synuclein-mediated toxicity in cellular models of Parkinson's disease (25
). The potential for SIRT2
modulation by SJW was investigated by Western blot analysis of SJW-treated MDA-MB-231 cells. SIRT2 protein levels decreased after 19 h of treatment with SJW (). Provided results similar to ours can be repeated in neuronal cells, the ability of SJW to deactivate SIRT2 may have implications for potential therapeutic use of SJW in treatment of Parkinson Disease, as SIRT2 inactivation has been found to restrict Lewy body formation and lead to the rescue of neuronal cells (25
It should be noted that the bioavailability of SJW compounds within the body and at physiological target sites will play an important role in the evaluation of potential mode of action models generated by genetic studies. Unfortunately, while many reports exist which describe the effect of SJW on the bioavailability of drugs taken concomitantly, few studies have been conducted to investigate the pharmacokinetic profile of SJW compounds in humans after oral ingestion. However, in the case of our suggestion herein that SJW may promote wound healing through the activation of angiogenic activities, our hypothesis is bolstered by the understanding that traditionally SJW salves were applied topically to wounds, which may allow SJW compounds direct access to target cells at the wound site.
In summary, we have shown that chemical-genetic profiling in yeast represents a powerful tool for the identification of human intracellular targets of dietary, bioactive botanical products, such as the medicinal plant SJW. We identified 78 genes previously undescribed as essential to the adaptive response to SJW. Fifty-two of these yeast genes have human orthologs, some of which have been implicated in neurological diseases, psychiatric disorders, angiogenesis, and human cancer. Three putative human intracellular targets predicted by chemical-genetic profiling in yeast (HIF1α, VEGF, and SIRT2) were confirmed by cell-based assays of SJW-treated human cells. Future research will seek to better understand the role of individual constituents of SJW in the adaptive responses observed using the whole SJW extract. We envision that the approach described here may be useful to the functional food and agricultural research community in the determination of molecular mechanisms of action for bioactive botanical products as whole extracts, the form in which they are most commonly used in traditional medicine, prior to the identification of singular bioactive constituents. Subsequently, individual compounds and/ or synergies among constituents that are responsible for various aspects of the overall molecular mechanism can be elucidated by comparison of individual chemogenomic profiles to that of the whole extract.