Anti-myeloma activity of the sesquiterpene lactone cnicin: impact on Pim-2 kinase as a novel therapeutic target
Karin Jöhrer,1 Marlene Obkircher,1 Daniel Neureiter,2 Johanna Parteli,1 Claudia Zelle-Rieser,1 Eva Maizner,1 Johann Kern,3 Martin Hermann,4 Frank Hamacher,5 Olaf Merkel,5 Nathalie Wacht,5 Christian Zidorn,6 Marcel Scheideler,7 and Richard Greil1,5
1Tyrolean Cancer Research Institute, Innrain 66, 6020 Innsbruck, Austria; 2Institute of Pathology, University Hospital Salzburg, Müllner Hauptstrasse 48, 5020 Salzburg, Austria; 3Dept of Internal Medicine V, Innsbruck Medical University; 4KMT Laboratory, Dept. of Visceral-, Transplant- and Thoracic Surgery, Innsbruck Medical University; 5Laboratory for Immunological and Molecular Cancer Research, Third Medical Department, University Hospital Salzburg; 6Institute of Pharmacy, Dept. of Pharmacognosy, University of Innsbruck; 7Institute for Genomics and Bioinformatics, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria;
Journal of Molecular Medicine 2012
Supplemental Figure and Table Legends
Suppl. Fig. 1 Impact of cnicin on myeloma cell death and cell cycle. a Flow cytometry analysis of NCI-H929 cells showing early (AnnexinV+/PI-) and late (AnnexinV+/PI+) apoptotic cells after 48hrs of cnicin-treatment. b Detailed time- and concentration courses of cnicin treatment using NCI-H929 cells. Percentages of cell survival compared to control are shown. c Propidium iodide staining of DNA was utilized to measure effects of cnicin-treatment on cell cycle phases in NCI-H929 cells (upper panel). Experimental data (n=3) is summarized as % of cells in specific cell cycle phases in the graph below. d Cell survival of NCI cells in response to cnicin (cni) and parthenolide (ptn) treatment.
Suppl. Fig. 2 Myeloma cell line NCI-H929 and different cell fractions (CD19+ B-cells, CD4+ and CD8+ T-cells) from PBMC of healthy donors (n=4) were treated with cnicin and cell survival was analyzed after 24hrs (flow cytometry analysis). b Beclin-1 expression in NCI-H929 cells was monitored in response to cnicin-treatment (2 and 5µM) over 6-24hrs by real-time PCR. Fold expression of control is depicted.
Suppl. Fig. 3 Effects of cnicin-treatment on activity of caspases, ROS, and NF-kB. a MM.1S cells were exposed to cnicin (2µM) for 3-12 hrs and cleavage of Caspases (Casp) 3, 8, 9 (FL=full length protein, CL=cleaved fragment) was analyzed. Actin served as loading control. b MM.1S cells were treated with indicated concentrations of cnicin for 24 and 48hrs +/- pan-caspase inhibitor Q-VD-OPH. Cell survival was measured by flow cytometry analysis (mean percentage +/- SD, n=3, *p<0.05, Students’ paired T-test). c Activation of ROS in MM.1S was analyzed by confocal microscopy after 1 h incubation with different concentrations of cnicin as indicated. ROS accumulation is visible as white spots within the cells. The effect of concomitant treatment with cnicin and ROS-inhibitors (NAC, 1 mM, DTT, 0.25 mM, n=3) on cell survival was measured after 48hrs as above. White bars indicate 10µm. d Expression of p-AKT and p-p38 in MM.1S cells was investigated by immunoblot at the indicated time points of cnicin treatment (2µM; actin serves as loading control).
Suppl. Fig. 4 Expression analyses of cnicin target-gene Pim-2 and functional analysis. a Constitutive Pim-2 expression in B cells of three healthy donors (HD 1-3) compared to myeloma cell lines NCI, MM.1S, and U266 is shown. Expression is depicted in arbitrary units normalized to HPRT. b Pim-2 protein is downregulated in response to cnicin treatment in MM.1S cells (2µM). c Comparison of spontaneous apoptosis rates of control siRNA (NTC) and Pim-2 siRNA-treated MM.1S cells 48 hrs past transfection (left graph; n=5, % cell survival +/-SD compared to control are displayed, p=0.0029, Students’ paired T-test). Right blots: Efficacy of Pim-2 knock-down in MM.1S cells analyzed by Western blot (NTC= non-targeting control). Actin is used as a loading control. d Pim-2 expression in MM.1S cells in response to IL-6 and cnicin treatment (6hrs, Real-time PCR, fold expression of control is shown, data normalized to HPRT).
Suppl. table 1 Detailed effects of combination treatments. Cell lines NCI-H929 and U266 were treated with combinations of a cnicin (cni, µM) and AKT-inhibitor VIII (akti, µM), b cnicin and melphalan (mel, µM), and c bortezomib (btz ng/ml), respectively and combination index values (CI) are shown. (Fa= fraction affected, equates to the percentage of AnnV+/PI+ and AnnV+/PI- cells; CI<0.9 display synergism, CI>1.1 mark antagonism, CI values of additive effects are between 0.9-1.1).
Suppl. table 2 Cnicin-induced gene regulation. Gene expression under cnicin treatment (6 hrs, NCI-H929 2µM, OPM-2 and U266 5µM) was measured by microarray analysis. Fold regulation by treatment is displayed (mean +/-SD).