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 expressionin NCI-H929 cellswas monitored in response to cnicin-treatment (2 and 5µM) over 6-24hrs by real-time PCR. Fold expression of control is depicted.
Suppl. Fig. 3Effects of cnicin-treatment on activity of caspases, ROS, and NF-B. 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 1h 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 ofcombination 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).