Interactions of echinacea and spilanthes with human metabolizing systems

"Increasing use of herbal products in recent years demand further studies relating to their safety and efficacy. Since cytochrome P450 enzymes play a major role in drug metabolism, studying their interactions with herbal extracts would be an important step in this direction. In the current study, Echinacea purpurea root and Spilanthes acmella whole flowering plant extracts were tested against CYP2C9, CYP1A2, CYP2A6 and CYP2E1. Both Echinacea and Spilanthes showed moderate inhibition against CYP2C9 while they both failed to show any inhibition towards CYP1A2. Echinacea showed moderate inhibition against both CYP2A6 and CYP2E1 but raw Spilanthes extract did not show any interaction with CYP2A6. It showed mild inhibition of CYP2E1 when used in high concentration. Isobutyl amides, known to be the only bioavailable components of Echinacea and Spilanthes were tested for their ability to inhibit CYP2E1. The four isobutyl amides present in major proportions in the ethanolic preparation of Echinacea purpurea root were isolated and shown to be potent inhibitors of CYP2E1. Spilanthol, the only major isobutyl amide found in the ethanolic preparation of Spilanthes acmella whole flowering plant was isolated and it showed strong inhibitory properties against CYP2E1."--Abstract from author supplied metadata

Effects of herbal products and their constituents on human cytochrome P4502E1 activity

Ethanolic extracts from fresh Echinacea purpurea and Spilanthes acmella and dried Hydrastis canadensis were examined with regard to their ability to inhibit cytochrome P4502E1 mediated oxidation of p-nitrophenol in vitro. In addition, individual constituents of these extracts, including alkylamides from E. purpurea and S. acmella, caffeic acid derivatives from E. purpurea, and several of the major alkaloids from H. canadensis, were tested for inhibition using the same assay. H. canadensis (goldenseal) was a strong inhibitor of the P4502E1, and the inhibition appeared to be related to the presence of the alkaloids berberine, hydrastine and canadine in the extract. These compounds inhibited 2E1 with KI values ranging from 2.8 µM for hydrastine to 18 µM for berberine. The alkylamides present in E. purpurea and S. acmella also showed significant inhibition at concentrations as low as 25 µM, whereas the caffeic acid derivatives had no effect. Commercial green tea preparations, along with four of the individual tea catechins, were also examined and were found to have no effect on the activity of P4502E1

Design of a Flexible Cell-Based Assay for the Evaluation of Heat Shock Protein 70 Expression Modulators

Heat shock protein 70 (Hsp70) is a chaperone protein that helps protect against cellular stress, a function that may be co-opted to fight human diseases. In particular, the upregulation of Hsp70 can suppress the neurotoxicity of misfolded proteins, suggesting possible therapeutic strategies in neurodegenerative diseases. Alternatively, in cancer cells where high levels of Hsp70 inhibit both intrinsic and extrinsic apoptotic pathways, a reduction in Hsp70 levels may induce apoptosis. To evaluate and identify, in a single assay format, small molecules that induce or inhibit endogenous Hsp70, we have designed and optimized a microtiter assay that relies on whole-cell immunodetection of Hsp70. The assay utilizes a minimal number of neuronal or cancer cells, yet is sufficiently sensitive and reproducible to permit quantitative determinations. We further validated the assay using a panel of Hsp70 modulators. In conclusion, we have developed an assay that is fast, robust, and cost efficient. As such, it can be implemented in most research laboratories. The assay should greatly improve the speed at which novel Hsp70 inducers and inhibitors of expression can be identified and evaluated

Abstract 3029: Biochemical evidence towards the existence of an oncogenic Hsp90 complex

Abstract To maintain homeostasis, cells employ intricate molecular machineries comprised of thousands of proteins programmed to execute well-defined functions. Dysregulation of these pathways, through protein mis-expression or mutation, provides biological advantages that confer the malignant phenotype. At the molecular level, this requires cells to invest energy in maintaining the stability and function of these proteins, and for this reason cancer cells co-opt molecular chaperones, including Hsp90. Hsp90 is recognized to play important roles in maintaining the transformed phenotype - the chaperone and its associated co-chaperones assist in the correct folding of cellular proteins, collectively referred to as “client proteins,” many of which are effectors of signal transduction pathways controlling cell growth, differentiation, the DNA damage response, and cell survival. Tumor cell addiction to these proteins (i.e. through mutations, aberrant expression, improper cellular translocation, etc) thus makes them critically reliant on Hsp90. While Hsp90 is expressed in all cells and tissues, it was shown that tumors preferentially contain Hsp90 that is in a higher order multi-chaperone complex with high affinity for certain Hsp90 inhibitors, while normal tissues harbor a latent, uncomplexed Hsp90 that has low affinity for these inhibitors. We here extend this model and propose that Hsp90 forms biochemically distinct complexes in cancer cells. In this view, a major fraction of cancer cell Hsp90 retains “house keeping” chaperone functions similar to normal cells, whereas a functionally distinct Hsp90 pool enriched or expanded in cancer cells specifically interacts with oncogenic proteins required to maintain tumor cell survival. Perhaps this Hsp90 fraction represents a cell stress specific form of chaperone complex that is expanded and constitutively maintained in the tumor cell context. Our data suggest that it may execute functions necessary to maintain the malignant phenotype. One such role is to regulate the folding of mutated (i.e. mB-Raf) or chimeric proteins (i.e. Bcr-Abl). We here also present experimental evidence for an additional role; that is, to facilitate scaffolding and complex formation of molecules involved in aberrantly activated signaling complexes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3029. doi:1538-7445.AM2012-302

Abstract 1263: Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90

Abstract Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1263. doi:1538-7445.AM2012-126

Selective compounds define Hsp90 as a major inhibitor of apoptosis in small-cell lung cancer

The heat shock protein 90 (Hsp90) has a critical role in malignant transformation. Whereas its ability to maintain the functional conformations of mutant and aberrant oncoproteins is established, a transformation-specific regulation of the antiapoptotic phenotype by Hsp90 is poorly understood. By using selective compounds, we have discovered that small-cell lung carcinoma is a distinctive cellular system in which apoptosis is mainly regulated by Hsp90. Unlike the well-characterized antiapoptotic chaperone Hsp70, Hsp90 is not a general inhibitor of apoptosis, but it assumes this role in systems such as small-cell lung carcinoma, in which apoptosis is uniquely dependent on and effected through the intrinsic pathway, without involvement of caspase elements upstream of mitochondria or alternate pathways that are not apoptosome- channeled. These results provide important evidence for a transformation- specific interplay between chaperones in regulating apoptosis in malignant cells. © 2007 Nature Publishing Group

Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell’s sensitivity to Hsp90 inhibition