Proteome Analysis Reveals a Distinct Molecular Profile of Cellular Stress Following Incubation of DDT₁-MF2 Smooth Muscle Cells in the Presence of a High Concentration of Hyperforin

Abstract

The acylphloroglucinol derivative hyperforin is a major constituent of St. John’s wort extracts (Hypericum perforatum L.), which has been demonstrated to contribute to the antidepressant action of this herbal drug. In previous investigations we observed that hyperforin causes a rapid stimulation of intracellular calcium mobilization and enhances extracellular acidification in the hamster vas deferens smooth muscle cell line DDT₁-MF2. To obtain further insight into its mode of action, we have now examined if these effects are accompanied by changes in protein expression. Cells were incubated with hyperforin for 15 min at a concentration of 1 μg/mL. Proteome analysis in cell lysates was accomplished by two-dimensional gel electrophoresis (2D-PAGE) and proteins were visualized by silver staining. Differences in the expression pattern between hyperforin- and vehicle-treated cells were displayed by computer-assisted differential display and identification of selected protein spots was performed by peptide mass fingerprinting after digestion with trypsin. Following incubation with hyperforin marked changes in the expression of several proteins were evident. A particularly strong change was observed for 6 proteins, which were identified as tubulin-beta, enolase 3, SYNCRIP, endoplasmin, elongation factor 2 and HSP84. As these proteins are known to be involved in cellular responses to stress by regulating energy metabolism as well as synthesis, intracellular transport and folding of proteins, our results suggest that the effects observed are not components of the normal pharmacological activity profile of hyperforin but are rather indicative of cellular stress promoting activity at higher concentrations.

Key words

Hyperforin - proteome analysis - stress response - DDT₁-MF1 cells - Hypericum perforatum - Clusiaceae

References

• 1 Di Carlo G, Borrelli F, Ernst E, Izzo A A. St John’s wort: Prozac from the plant kingdom.  Trends Pharmacol Sci. 2001;  22 292-7
• 2 Schempp C M, Pelz K, Wittmer A, Schopf E, Simon J C. Antibacterial activity of hyperforin from St John’s wort, against multiresistant Staphylococcus aureus and Gram-positive bacteria.  Lancet. 1999;  353 2129
• 3 Albert D, Zündorf I, Dingermann T, Müller W E, Steinhilber D, Werz O. Hyperforin is a dual inhibitor of cyclooxygenase-1 and 5-lipoxygenase.  Biochem Pharmacol. 2002;  64 1767-75
• 4 Chatterjee S S, Nöldner M, Koch E, Erdelmeier C. Antidepressant activity of Hypericum perforatum and hyperforin: the neglected possibility.  Pharmacopsychiatry. 1998;  31 (Suppl 1) 7-15
• 5 Koch E, Chatterjee S S. Hyperforin stimulates intracellular calcium mobilisation and enhances extracellular acidification in DDT1-MF2 smooth muscle cells.  Pharmcopsychiatry. 2001;  34 (Suppl. 1) 70-3
• 6 Kennedy S. The role of proteomics in toxicology: identification of biomarkers of toxicity by protein expression analysis.  Biomarkers. 2002;  7 269-90
• 7 Šosˇkic V, Görlach M, Poznanovic S, Boehmer F D, Godovac-Zimmermann J. Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor.  Biochemistry. 1999;  38 1757-64
• 8 Banks R E, Dunn M J, Hochstrasser D F, Sanchez J C, Blackstock W, Pappin D J, Selby P J. Proteomics: new perspectives, new biomedical opportunities.  Lancet. 2000;  356 1749-56
• 9 Walss-Bass C, Prasad V, Kreisberg J I, Luduena R F. Interaction of the beta-IV-tubulin isotype with actin stress fibers in cultured rat kidney mesangial cells.  Cell Motil Cytoskeleton. 2001;  49 200-7
• 10 Guha S, Manna T K, Das K P, Bhattacharyya B. Chaperone-like activity of tubulin.  J Biol Chem. 1998;  273 30 077-80
• 11 Larsen M R, Larsen P M, Fey S J, Roepstorff P. Characterization of differently processed forms of enolase 2 from Saccharomyces cerevisiae by two-dimensional gel electrophoresis and mass spectrometry.  Electrophoresis. 2001;  22 566-75
• 12 Roth E J r. Plasmodium falciparum carbohydrate metabolism: a connection between host cell and parasite.  Blood Cells. 1990;  16 453-60
• 13 Mizutani A, Fukuda M, Ibata K, Shiraishi Y, Mikoshiba K. SYNCRIP, a cytoplasmic counterpart of heterogeneous nuclear ribonucleoprotein R, interacts with ubiquitous synaptotagmin isoforms.  J Biol Chem. 2000;  275 9823-31
• 14 Harris C E, Boden R A, Astell C R. A novel heterogeneous nuclear ribonucleoprotein-like protein interacts with NS1 of the minute virus of mice.  J Virol. 1999;  73 72-80
• 15 Gerner C, Frohwein U, Gotzmann J, Bayer E, Gelbmann D, Bursch W, Schulte-Hermann R. The Fas-induced apoptosis analyzed by high throughput proteome analysis.  J Biol Chem. 2000;  275 39 018-26
• 16 Riera M, Roher N, Miro F, Gil C, Trujillo R, Aguilera J, Plana M, Itarte E. Association of protein kinase CK2 with eukaryotic translation initiation factor eIF-2 and with grp94/endoplasmin.  Mol Cell Biochem. 1999;  191 97-104
• 17 Piwien-Pilipuk G, Ayala A, Machado A, Galigniana M D. Impairment of mineralocorticoid receptor (MR)-dependent biological response by oxidative stress and aging: correlation with post-translational modification of MR and decreased ADP-ribosylatable level of elongating factor 2 in kidney cells.  J Biol Chem. 2002;  277 11 896-903
• 18 Kang K R, Lee S Y. Effect of serum and hydrogen peroxide on the Ca²⁺/calmodulin-dependent phosphorylation of eukaryotic elongation factor 2 (eEF-2) in Chinese hamster ovary cells.  Exp Mol Med. 2001;  33 98-204
• 19 Richter-Landsberg C, Goldbaum O. Stress proteins in neural cells: functional roles in health and disease.  Cell Mol Life Sci. 2003;  60 337-49
• 20 Ko H S, Uehara T, Nomura Y. Role of ubiquilin associated with protein-disulfide isomerase in the endoplasmic reticulum in stress-induced apoptotic cell death.  J Biol Chem. 2002;  277 35 386-92
• 21 Biber A, Fischer H, Römer A, Chatterjee S S. Oral bioavailability of hyperforin from Hypericum extracts in rats and human volunteers.  Pharmacopsychiatry. 1998;  31 (Suppl. 1) 36-43