Differential activities of thalidomide and isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells

Abstract

Thalidomide has emerged as an effective agent for treating multiple myeloma, however the precise mechanism of action remains unknown. Agents known to target the isoprenoid biosynthetic pathway (IBP) can have cytotoxic effects in myeloma cells. The interactions between thalidomide and IBP inhibitors in human multiple myeloma cells were evaluated. Enhanced cytotoxicity and induction of apoptosis were observed in RPMI-8226 cells. Examination of intracellular levels of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) revealed a wide variance in basal levels and response to IBP inhibitors. These findings provide a mechanism for the differential sensitivity of myeloma cells to pharmacologic manipulation of the IBP.

Figure 1

The isoprenoid biosynthetic pathway (IBP) and pharmacological inhibitors. Substrates and products of the pathway are shown in black, enzymes are shown in blue, and specific inhibitors are shown in red.

Figure 2

The cytotoxic effects of thalidomide and IBP inhibitors in myeloma cells. Cells (RPMI-8226 (A), H929 (B), U266 (C) were incubated with equimolar concentrations of drugs (lovastatin, Lov; GGTI-286, GGTI) for 48 hours prior to the addition of MTT. Data are expressed as a percentage of control. The * denotes p<0.05 and ** denotes p<0.01 per unpaired two-tailed t-test comparing thalidomide + IBP inhibitor to IBP inhibitor alone.

Figure 3

The effect of thalidomide and IBP inhibitors on induction of apoptosis in myeloma cells. Annexin V, propidium iodide (PI) flow cytometric experiments were performed in RPMI-8226 (A), H929 (B), and U266 (C) cells. Cells were incubated for 48 hours. The percentages of cells in the early apoptotic (Annexin V positive, PI negative (Ann+ PI-)) and late apoptotic/necrotic (Annexin V positive, PI positive (Ann+ PI+)) fractions are shown (n =3). * denotes p<0.05 per unpaired two-tailed t-test comparing thalidomide + IBP inhibitor to IBP inhibitor alone. D) Immunoblots depicting PARP cleavage. Cells were incubated for 48 hours with combinations of thalidomide (Thal, 50 μM), lovastatin (Lov, 50 μM) or DGBP (50 μM). The “*” denotes the C-terminal PARP cleavage product. β-Tubulin is shown as a loading control.

Figure 4

Add-back of select IBP intermediates prevents thalidomide/IBP inhibitor-induced cytotoxicity (A) and apoptosis (B) in RPMI-8226 cells. Cells were incubated for 48 hours in the presence or absence of thalidomide (10 μM) + IBP inhibitors (10 μM lovastatin or 10 μM DGBP) and IBP intermediates (1 mM mevalonate (Mev), 10 μM FPP, or 10 μM GGPP) prior to addition of MTT (A) or staining with Annexin V/PI (B). For the MTT assay, data are expressed as a percentage of control (n=4; mean ± standard error) and for the Annexin V/PI assay, data are expressed as the percentage of all Annexin-positive cells (n=2).

Figure 5

Effects of IBP inhibitors on protein prenylation in myeloma cells. Cells were incubated for 24 hours in the presence or absence of drugs. The Rap1a antibody recognizes only the unmodified form of Rap1a. β-Tubulin is shown as a loading control.

Figure 6

Effect of IBP inhibitors on FPP and GGPP levels in myeloma cells. Cells were incubated for 24 hours in the presence or absence of drugs. Data are presented as mean and standard deviation (n = 2-4 independent experiments).