Zoledronic acid induces antiproliferative and apoptotic effects in human pancreatic cancer cells in vitro

Abstract

Bisphosphonates (BPs) are an emerging class of drugs mostly used in the palliative care of cancer patients. We investigated the in vitro activity of the most potent antiresorptive BP, zoledronic acid (ZOL), on the growth and survival of three human pancreatic cancer (PC) cell lines (BxPC-3, CFPAC-1 and PANC-1). Pancreatic cancer frequently has a dysregulated p21(ras) pathway and therefore appears to be a suitable target for BPs that interfere with the prenylation of small GTP-binding proteins such as p21(ras). We found that ZOL induces growth inhibition (IC(50):10-50 micro M) and apoptotic death of PC cells. The proapoptotic effect was correlated to cleavage/activation of caspase-9 and poly(ADP)-ribose polymerase, but not of caspase-3. Moreover, we studied the p21(ras) signalling in cells exposed to ZOL and detected a reduction of p21(ras) and Raf-1 content and functional downregulation of the terminal enzyme ERK/MAPkinase and of the pKB/Akt survival pathway. Finally, we observed that ZOL induces significant cytoskeletal rearrangements. In conclusion, we demonstrated that ZOL induces growth inhibition and apoptosis on PC cells and interferes with growth and survival pathways downstream to p21(ras). These findings might be relevant for expanding application of BPs in cancer treatment.

Figure 1

Zoledronic acid inhibits the growth of PC cells. (A) The cell lines BxPC-3, CFPAC-1 and PANC-1 (3 × 10⁴ ml⁻¹) were seeded in 96-well plates and incubated for 24 h. After 24 h, medium was removed and replaced with medium containing increasing concentrations of ZOL (1–100 μM). After 72 h of incubation, cell proliferation was determined by the MTT assay. A value of 100% was assigned to untreated control cultures, and the antiproliferative effects of ZOL were calculated as percentage of residual growth. Each point is the mean value of at least four replicate experiments ±s.e. (B) The IC₅₀ drug concentration for each cell line was measured by a logarithmic interpolate curve on the percentage of residual growth at each concentration point (C and D) Morphological changes induced in cell cultures after 72 h exposure to ZOL. Analysis was performed by inverted phase-contrast microscopy. (C) Untreated cultures; (D) treated cultures with ZOL (50 μM).

Figure 2

(A) Apoptotic effects induced by ZOL on CFPAC-1, PANC-1, BxPC-3 PC cancer cells. The analysis of apoptosis was performed after exposure to 50 μM of ZOL by flow cytometric detection of Annexin-V immunostaining. Cells were pulse exposed for 30, 60 and 180 min or continuously exposed to the drug. The PI method was used when apoptosis was evaluated 24 h after beginning treatment. Data are expressed as the mean value of at least four replicate experiments ±s.e. (B) Flow cytometric profiles from a representative experiment performed on CFPAC-1 cells. The percentage of apoptotic stained cells (%) is indicated in each quadrant.

Figure 3

Role of caspase-9/-3 and PARP in apoptotic cell death induced by ZOL. (A) Detection of caspase-9/-3 and PARP in PC cells after ZOL treatment. Western blotting analysis was performed on protein extracts from solubilized whole cell pellets from BxPC-3 and PANC1 PC cell lines after 48 h exposure to ZOL (50 μM) demonstrating abundant cleaved species of both caspase-9 and PARP in lysates for drug-exposed cells, while caspase-3 remained almost completely uncleaved. The experiments were performed at least three times and the results were always similar. (B) Apoptotic effects induced by ZOL in the presence of caspase inhibitors. Analysis was performed by flow cytometric immunodetection of Annexin-V staining on BxPC-3 cells after 48 h exposure to ZOL (50 μM). VAD is a caspase-9 inhibitor, VEID is a specific caspase-6 inhibitor and DEVD is a caspase-3 inhibitor. Pretreatment with 15 μM VAD and 15 μM VEID significantly antagonised ZOL-induced apoptosis (P<0.005).

Figure 4

Effects of ZOL treatment on p21^ras/Raf-1/MEK1/ERK signalling and PK-B/Akt pathways in PC cells. Western blotting analysis of p21^ras (A), raf-1 (B), Erk-1/2 (D) and pErk (E). The immunoblotting shows downregulation of p21^ras (A) and raf-1 (B) levels after 48 h of exposure to 50 μM ZOL. While no significant effects were detected on Erk-1/2 expression after exposure of cells to the drug (D), the active phosphorylated Erk-1/2 (recognised by an anti-pERK Mab) levels were greatly reduced in ZOL-treated cells (E). (F and G) Akt expression and functional analysis in BxPC-3, PANC-1 and CFPAC-1 pancreatic adenocarcinoma cells. For detection of Akt activity, immunoprecipitation was performed from cell lysates with anti-Akt antibody, and an in vitro kinase assay was performed using GSK3α/β as a substrate, followed by Western blotting analysis with antiphospho-GSK3α/β. GSK3α/β phosphorylation by immunoprecipitated Akt was downregulated after 48 h treatment of PC cells with 50 μM ZOL (F), whereas the Akt protein levels remained unmodified. The experiments were performed at least three times and the results were always similar. CTR: untreated cells; ZOL: ZOL-treated cells.

Figure 5

Analysis of cytoskeletal reorganisation by confocal microscopy on PANC-1 PC cells after 48 h exposure to ZOL (15 μM). The figure shows actin architecture rearrangements in cortical rings. The cells were examined under a confocal microscope at a magnification of × 100. The experiments were performed at least three times and the results were always similar.