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Blood, Vol. 113, Issue 17, 4027-4037, April 23, 2009
Identification of a potent natural triterpenoid inhibitor of proteosome chymotrypsin-like activity and NF- B with antimyeloma activity in vitro and in vivo
Blood Tiedemann et al.
113: 4027
Supplemental materials for: Tiedemann et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 66.5 KB)
- Figure S1. Pristimerin administered to cells inhibits proteosome enzymatic activity (JPG, 45.9 KB)
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H929 myeloma cells were treated with pristimerin at various concentrations (A) or with pristimerin 500nM for various durations (B) and were then harvested by centrifugation and lysed. Proteosome enzymatic activity present in the lysates of treated cells was assessed by the degradation of enzyme-specific fluorogenic peptide substrates at 37°C over 4 hours. In this assay, non-proteosomal peptidase activity may contribute to background non-responsive chymotrypsin-like activity (~30% residual chymotrypsin-like activity is detected with both pristimerin and bortezomib); however, despite this, specific and potent inhibition of cell-based chymotrypsin-like activity by prisitimerin is demonstrated. The effect of pristimerin on proteosome activity of cells prior to lysis was additionally assessed by immunoblot analysis of H929 lysates for accumulation of ubiquitinated protein (as shown in Fig. 4C, D).
- Figure S2. Prisitimerin causes increased cellular ubiquitinated protein and inhibits I κB phosphorylation (JPG, 69.9 KB)
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H929 myeloma cells, untreated, or treated with (A) pristimerin 500nM (~3× IC50), bortezomib 10nM (3–5× IC50), melphalan 10uM (~3× IC50) or (B) bortezomib 100nM (30–50 × IC50) were harvested, lysed and assessed by immunoblot. Phosphorylated or ubiqintated IkB levels are assessed with respective short (lower panel) or long (middle panel) exposure times. Pristimerin caused early accumulation of cellular ubiquitinated protein at 1.5h that was pronounced at 6h and comparable to the response observed with bortezomib 100nM. Both pristimerin (at 1.5h) and bortezomib were associated with smearing of IκB staining (associated with gain in molecular size) corresponding to ubiquitinated IκB species, though longer (6h) treatment of cells with pristimerin, but not bortezomib, was followed by loss of IκB phosphorylation and loss of phosphorylation-driven ubiquitination (as shown by relative accumulation of the lower molecular size IκB band). As the L35A5 antibody used for IκB staining is directed against the unmodified N-terminus of IκB and as IκB is modified by ubiquitination on its N-terminus (on L21, L22), the affinity of L35A5 for ubiquitinated IκB, and the extent of staining of ubiquitinated IκB species compared to unmodified IκB is likely to be less than linear. Melphalan at 10µM concentration caused minimal accumulation of ubiquitinated protein at a comparable time-point and like bortezomib, appeared to increase rather than decrease IκB phosphorylation.
- Figure S3. Pristimerin is selectively cytotoxic to primary myeloma cells in mixed-lineage bone marrow cultures (JPG, 90.4 KB)
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Flow cytometric data from Fig. 5 analyzed and presented as normalized viability (normalized to the viability of untreated samples), by CD138 compartment and pristimerin dose, showing preferential loss of CD138+ tumor cells with pristimerin 100nM or 200nM, relative to any loss of CD138-negative cells.
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