The inhibitory effect of alendronate, a nitrogen-containing bisphosphonate on the PI3K-Akt-NFkappaB pathway in osteosarcoma cells

Abstract

1 Bisphosphonates are inhibitors of tumor cell growth as well as of bone resorption by inducing cell apoptosis. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. The aim of the present study was to determine the effect of alendronate, one of the nitrogen-containing bisphosphonates on the phoshoinositide 3-kinase (PI3K)-Akt-NFkappaB pathway, the major cell survival pathway. 2 The PI3K-Akt-NFkappaB pathway was activated in the osteosarcoma cell line MG-63 treated with tumor necrosis factor-alpha or insulin. Saos-2 was also used in some experiments. This was assessed by the production of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), increased PI3K activity, phosphorylation of Akt at serine 473 and threonine 308, increase in activity of the inhibitor of nuclear factor kappaB (IkappaB) kinase (IKK) and finally phosphorylation of IkappaB and its subsequent degradation. 3 Pretreatment with alendronate at 100 microM for 24 h prior to the stimulation with tumor necrosis factor-alpha or insulin partially inhibited the IkappaB phosphorylation and degradation. These events were more clearly observed in the presence of inhibitors of proteasomes, which are responsible for the degradation of IkappaB. The drug also partially inhibited the activity of IKK, but almost fully inhibited the phosphorylation of Akt and the production of PtdIns(3,4,5)P(3). 4 The inhibitory effect of alendronate on IkappaB phosphorylation and degradation was not attenuated by the exogenous addition of geranylgeraniol to replenish the cytosolic isoprenyl lipid substrate. 5 The present findings demonstrate that alendronate inhibited the PI3K-Akt-NFkappaB cell survival pathway at the point of PI3K activation, thus indicating the presence of new targets of alendronate.

Figure 1

Phosphorylation and degradation of IκB. (a) MG-63 cells (30 × 10⁴) were starved for 12 h and then treated with or without 100 μM alendronate for 24 h, followed by the stimulation with 5 ng ml⁻¹ TNFα for 1, 3, 5, 7 and 10 min. Cell lysates were analyzed by immunoblotting using polyclonal anti-phospho-IκB-α (Ser32) antibody (Cell Signaling Technology, Inc.) and polyclonal anti-specific-IκB-α (Cell Signaling Technology, Inc.). (b) MG-63 cells (30 × 10⁴) were starved for 12 h and incubated with 10 μM lactacystin for 2 h, followed by treatment with 100 μM alendronate for 24 h and then 5 ng ml⁻¹ TNFα for 1, 3, 5, 7, 10 and 15 min. Cell lysates were analyzed by immunoblotting as described above. Upper panels and lower graphs indicate typical immunoblots and a summary of five separate experiments shown as the mean±s.e., respectively. ^*Indicates a significant difference when compared with that seen in the control cells at the same time point. (c) MG-63 cells were treated as described in (a), followed by stimulation with 5 ng ml⁻¹ TNFα or 10 ng ml⁻¹ insulin for the period indicated. Cell lysates were immunoblotted as described above. The blot shown is typical of three experiments.

Figure 2

Activity of IKK. MG-63 cells (1 × 10⁶) were starved for 12 h and treated with or without 100 μM alendronate for 24 h, followed by stimulation with 5 ng ml⁻¹ TNFα for the period indicated. Lysates were immunoprecipitated by polyclonal anti-IKKβ antibody (Santa Cruz Biotechnology, Inc.). The procedures used are described in ‘Methods'. Upper panels indicate typical immunoblots, and the lower graph indicates the results expressed as the density of anti-phospho-IκB relative to that of anti-IKKβ in the mean±s.e. of five independent experiments. ^*Indicates a significant difference when compared with that seen in the control cells at the same time point.

Figure 3

Phosphorylation of Akt. MG-63 cells (30 × 10⁴) were starved for 12 h and treated with or without 100 μM alendronate for 24 h, followed by stimulation with 5 ng ml⁻¹ TNFα for the period indicated. Cell lysates were analyzed by immunoblotting using polyclonal anti-phospho-Akt (Ser473) (New England BioLabs Inc.) or polyclonal anti-phospho-Akt (Thr308) (New England BioLabs Inc.) for the phosphorylation assay, and polyclonal anti-Akt (New England BioLabs Inc.) for the quantification. The upper panel and lower graph show typical immunoblots and the results are expressed as the density of anti-phospho-Akt relative to that of anti-Akt and shown as the mean±s.e. of five independent experiments, respectively. ^*Indicates a significant difference when compared with that seen in the control cells at the same time point.

Figure 4

Activity of PI3K. MG-63 cells (1 × 10⁶) were starved for 12 h and treated with or without 100 μM alendronate for 24 h or 25 μM LY294002 for 2 h, followed by incubation with [³²P]orthophosphate (125 μCi ml⁻¹) for 3 h at 37°C. After extensive washing, cells were stimulated with 5 ng ml⁻¹ TNFα or 10 ng ml⁻¹ insulin for 10 min. PtdIns(3,4,5)P₃ production assay was performed as described in ‘Methods'. Similar results were seen in four other experiments.

Figure 5

Radio ligand binding assay. MG-63 cells (1 × 10⁴ cells in a well) were starved for 12 h in 96-well plates (CulturPlate-96) and treated with or without 100 μM alendronate for 24 h. After washing with a phosphate-buffered saline three times, cells were incubated with 0.2, 0.5, 1.0, 2.0, 3.0, 4.0 or 5.0 nM [¹²⁵I]TNFα (Amersham Bioscience) for 2 h at 4°C. After washing with PBS three times, Microscint™ 40 (Packard) was added to each well, followed by scintillation counting using Top Count NXT™ (Perkin-Elmer). Open and closed symbols represent the specific binding obtained with control and alendronate-treated cells, respectively. Nonspecific binding assayed in the presence of 1000-fold excess amount of unlabeled TNFα was within 150–300 c.p.m. Each point indicates the mean±s.e. of six independent measurements.

Figure 6

Experiments for cell death. MG-63 cells (30 × 10⁴ cells) were cultured as described above, followed by starvation for 12 h, with or without TNFα at 5 ng ml⁻¹. Alendronate (100 μM), with or without GGOH (20 μM), was then added for 24 h, either in the presence or absence of TNFα. Cells attached to dishes were collected and then viability was assessed using the Trypan blue exclusion test. ^*Indicates a significant difference from the control cells.

Figure 7

The effect of geranylgeraniol on phosphorylation and degradation of IκB. MG-63 cells starved for 12 h were treated with alendronate with or without GGOH (20 μM) for 24 h, followed by stimulation with 5 ng ml⁻¹ TNFα for 3 or 5 min. Cell lysates were immunoblotted as described above. The blot shown is typical of three experiments.