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Zoledronic acid treatment impairs protein geranyl-geranylation for biological effects in prostatic cells
© Goffinet et al; licensee BioMed Central Ltd. 2006
Received: 02 February 2006
Accepted: 15 March 2006
Published: 15 March 2006
Nitrogen-containing bisphosphonates (N-BPs) have been designed to inhibit osteoclast-mediated bone resorption. However, it is now accepted that part of their anti-tumor activities is related to interference with the mevalonate pathway.
We investigated the effects of zoledronic acid (ZOL), on cell proliferation and protein isoprenylation in two tumoral (LnCAP, PC-3,), and one normal established (PNT1-A) prostatic cell line. To assess if inhibition of geranyl-geranylation by ZOL impairs the biological activity of RhoA GTPase, we studied the LPA-induced formation of stress fibers. The inhibitory effect of ZOL on geranyl geranyl transferase I was checked biochemically. Activity of ZOL on cholesterol biosynthesis was determined by measuring the incorporation of 14C mevalonate in cholesterol.
ZOL induced dose-dependent inhibition of proliferation of all the three cell lines although it appeared more efficient on the untransformed PNT1A. Whatever the cell line, 20 μM ZOL-induced inhibition was reversed by geranyl-geraniol (GGOH) but neither by farnesol nor mevalonate. After 48 hours treatment of cells with 20 μM ZOL, geranyl-geranylation of Rap1A was abolished whereas farnesylation of HDJ-2 was unaffected. Inhibition of Rap1A geranyl-geranylation by ZOL was rescued by GGOH and not by FOH. Indeed, as observed with treatment by a geranyl-geranyl transferase inhibitor, treatment of PNT1-A cells with 20 μM ZOL prevented the LPA-induced formation of stress fibers. We checked that in vitro ZOL did not inhibit geranyl-geranyl-transferase I. ZOL strongly inhibited cholesterol biosynthesis up to 24 hours but at 48 hours 90% of this biosynthesis was rescued.
Although zoledronic acid is currently the most efficient bisphosphonate in metastatic prostate cancer management, its mechanism of action in prostatic cells remains unclear. We suggest in this work that although in first intention ZOL inhibits FPPsynthase its main biological actitivity is directed against protein Geranylgeranylation.
Bisphosphonates (BPs) have been used in oncological practice for many years to reduce skeletal complications and pain especially during myeloma and the metastatic phase of breast and prostate cancers. For many years their specific bone targeting suggested that the main mechanism of action of BPs was inhibition of bone resorption by direct effects on osteoclasts or other bone cells in the immediate microenvironment of osteoclasts. The late-generation BPs have a nitrogen-containing aliphatic side chain (pamidronate, alendronate, ibandronate) or heterocyclic ring (zoledronate). Their anti-resorptive potency is up to 1000-fold greater than that of non-amino BPs and they exert their cellular effects by interference with the mevalonate (MVA) pathway . Many studies have investigated the nature of the enzymatic targets of N-BPs within the MVA pathway. In vitro, squalene synthase activity was shown to be affected by some N-BPs but neither by pamidronate nor alendronate. More recently, farnesyl-pyrophosphate (FPP) synthase was proposed as the main enzymatic target of alendronate, risedronate, ibandronate, and pamidronate in vitro [4–6]. Some authors suggested that isopentenyl (IPP) isomerase could also be inhibited by novel N-BPs [4, 5]. In vivo, some studies confirmed the hypothesis of an inhibition of FPP synthase because apoptosis [7–9] and caspase activation  induced by several N-BPs were reversed by addition of FPP and GGPP or of cell-permeable analogs, farnesol (FOH) and geranyl-geraniol (GGOH). Conversely, recent in vivo studies suggested that the inhibition of the mevalonate pathway by N-BPs lies downstream the FPP synthesis step; indeed, several cellular effects of N-BPs such as inhibition of osteoclast formation, inhibition of ovarian cancer cell migration  or inhibition of breast cancer cell invasion  were reversed only by GGOH but not by FOH. Whatever the enzymatic target(s) of N-BPs most previous reports agree to suggest that the action of N-BPs results in the impairment of protein isoprenylation in osteoclasts or bone explants [4, 5, 8, 10, 13, 14] as well as in tumoral cell lines [9, 12, 15–17]. It remains unclear whether protein farnesylation and geranyl-geranylation are equally affected by N-BPs. Some recent data suggest that geranyl-geranylation, especially of Rho GTPases, may be the main target of their anti-invasive effect [11, 12] although their apoptotic effect may be related to the inhibition of Ras farnesylation [16, 17].
In prostate tumoral cells, few studies have investigated the cellular or molecular effects of N-BPs, while zoledronate (ZOL) and pamidronate (PAM) are widely used to prevent skeletal complications of prostate cancer and of androgen deprivation therapy [18, 19]. After the report of the inhibition of prostate carcinoma cell invasion by several N-BPs, including ZOL , PAM and ZOL were shown to inhibit growth in three widely used prostatic cell lines, PC-3, DU-145 and LNCaP . The anti-tumor effect of alendronate, ZOL and PAM was correlated to their inhibition of the MWA pathway in prostate cells [15, 17], favoring the hypothesis of an inhibition of FPP synthesis, protein geranyl-geranylation was not assessed although GGOH appeared more effective than FOH to rescue ZOL-induced apoptosis.
In the present work, we investigated i) the effect of ZOL and MVA-derived metabolites on tumoral and normal immortalized prostatic cell line proliferation; ii) the modification by ZOL of the prenylation status of farnesylated (HDJ-2) and geranyl-geranylated (Rap1A) proteins in prostatic cells; iii) the impact of ZOL on the biological activity of a geranyl-geranylated protein, RhoA; vi) the effect of ZOL on cholesterol biosynthesis.
Chemicals and materials
All material for cell culture was from Dutcher (Brumath, France). Growth media were purchased from Cambrex Bio Science (Verviers, Belgium) and fetal calf serum (FCS) from Invitrogen (Cergy-Pontoise, France). Zoledronic acid [hydrated disodium salt of 2-(imidazol-1-yl)-hydroxyl-ethylene-1,1-bisphosphonic acid)] was supplied by Novartis Pharma AG (Basel, Switzerland). Stock solutions (4 mM) were prepared in phosphate buffered saline (PBS) and aliquots were stored at -20°C to be diluted in culture medium prior to experiments. Peptidomimetic prenyl-transferase inhibitors FTI 277 and GGTI 298 [22, 23] were a generous gift from Pr S. Sebti (Tampa, Florida). Stock solutions (100 mM) were prepared in DMSO-10 mM DTT and aliquots were stored at -20°C to be diluted in culture medium prior to experiments. Y27632 was purchased from Calbiochem (La Jolla, California). Mevalonate (stock = MVA 200 mM), trans-transfarnesol (FOH; stock solutions 100 mM in ethanol, stored at -20°C), geranyl-geraniol (GGOH; stock solutions 100 mM in ethanol stored at -20°C), squalene (stock solutions 100 mM in ethanol stored at -20°C FOH), Cholesterol, L-α- lysophosphatidic acid (LPA) and paraformaldehyde (PFA) were purchased from Sigma Aldrich (St Quentin Fallavier, France). [14C]-mevalolactone (56Ci/mmol. specific activity) was from Perkin-Elmer.
Prostatic cell lines
LNCaP cells are prostatic tumoral human cells expressing an active but mutated (T877A) androgen receptor. Under androgenic stimulation, these cells produce prostate-specific antigen (PSA). PC-3 are prostatic tumoral human cells, which are non-responsive to androgenic stimulation. The PNT1-A cell line was developed by Cussenot et al. . The cells are normal epithelial cells immortalized by SV-40. They express the wild-type androgen receptor but, under androgenic stimulation in vitro, they do not secrete PSA into the culture medium. The three cell lines were purchased from the European Collection of Cell Culture (PC-3: # 90112714; LNCaP clone FCG: # 89110211; PNT1A: #95012614).
Prostatic cell lines were routinely grown either in RPMI 7% FCS (PC-3 and PNT1-A) or in RPMI 10% FCS supplemented by 10 mM Hepes and 1 mM sodium pyruvate (LNCaP). All cells were incubated at 37°C in a humidified 5% CO2 incubator.
Proliferation colorimetric assay
Proliferation was assessed by a colorimetric assay with sulforhodamine as described by Skehan . Cells were seeded in 96-well microtiter plates to finally obtain 80% confluence in control wells. At the end of the treatment period, the cells were fixed with TCA (1 h, 4°C) and then stained with 0.4 % sulforhodamine. After four washes with 1% acetic acid to remove unbound dye, the plates were dried and protein-bound dye was extracted with Tris (10 mM, pH = 10.5). Coloration was quantified at 540 nm in a microtiter plate (Multiskan® Multisoft, Labsystems)
Prenylation status analysis
Cells were scraped off then lysed with 150 μL of lysis buffer [20 mM Tris-HCl, 100 mM NaCl, 1% Triton X-100, 10 mM MgCl2, 5 mM EDTA, 20 mM NaF, 10 mM PNPP, 2 mM Na orthovanadate, and a cocktail of protease inhibitors (Sigma, France, final dilution 1/100). Proteins were quantified in cellular extracts by a Bradford assay. Five to forty μg of cleared protein extracts were separated on a 12.5% SDS-polyacrylamide gel and then transferred to a Hybond-P polyvinyldifluoride membrane (Amersham Biosciences). After pre-incubation with TBST (25 mM Tris, 140 mM NaCl, 0.1% Tween 20, pH 8) supplemented with 5% fat-free milk, the membranes were incubated overnight at 4°C with the primary antibody diluted in TBST 1 % milk. After three washes with TBST, the membranes were incubated for 1 hour at room temperature with the secondary antibody labeled by peroxidase. The antibody was visualized using the ECL Plus system (Amersham Biosciences) and luminescence quantified with Phophorimager® (Molecular Dynamics).
The following primary antibodies were used: anti-HDJ2 Ab-1 mouse monoclonal antibody (Interchim; clone KA2A5.6, diluted 1/10000), anti-prenylated Rap1A C17 goat polyclonal antibody (TEBU; ref SC-1482, diluted 1/2000), anti-total Rap1A-Krev1 rabbit polyclonal antibody (TEBU; ref SC-65, diluted1/1000). The following peroxidase-labeled secondary antibodies were used, all diluted 1/10000: anti-mouse (Biorad, ref 170–6516) anti-goat (Santa Cruz, ref sc-2033) or anti-rabbit (Biorad, ref 170–6515).
Visualization of actin cytoskeleton by fluorescence microscopy
At day 0 (D0), PNT1-A cells were seeded onto glass coverslips in 6-well plates to obtain 60% confluence on day 3 (D3). On day 1 (D1), the cells were serum-starved until treatment on D3. After exposure to different drugs and then after stimulation by LPA (20 μM, 5 hours on D3), the cells were fixed with 3% paraformaldehyde/PBS for 20 minutes and then permeabilized with 0.1% Triton- X-100/PBS for 5 minutes. To visualize the actin fibers, the coverslips were incubated with tetramethylrhodamine isothiocyanate-labeled phalloidin (Molecular Probes) for 30 minutes at room temperature. The cells were viewed on a Zeiss Axiophot microscope and pictures taken with a Princeton camera.
In vitro farnesyl-transferase (FTase) and geranyl-geranyl-transferase I (GGTase I) activity measurement
To investigate the putative inhibitory effect of ZOL on prenyl-transferase activity, in vitro prenylation assays were performed. As substrate proteins, we used bacterially expressed wild-type H-Ras protein for FTase assay and CVLL-H-Ras mutant protein (geranyl-geranylated form) for GGTase I assay. 1 μM substrate proteins were incubated with [3H]-prenyl pyrophosphate (0.25 μM final concentration, specific activity 8–10 Ci/mmol, American Radiolabeled Chemicals) and either recombinant FTase or recombinant GGTase I (50 ng) in a reaction medium containing 50 mM Tris, pH 7.7, 20 mM KCl, 10 mM MgCl2, 2 mM DTT, and 5 μM ZnCl2. The reaction volume was 50 μl. Reactions proceeded for 10 min at 30°C and were stopped by the addition of 0.5 ml of 4% SDS. Total protein was precipitated by the addition of 0.5 mL of 30% trichloroacetic acid. After 20 min, samples were filtered through 25-mm glass fiber filters (Schleicher & Schuell), which bound the prenylated protein leaving unprenylated protein and excess prenyl groups to go through the filter. Reaction tubes were washed with 2 × 2 mL of 4% SDS plus 6% trichloroacetic acid, and the washes were added to the filters. Bound protein was washed with a further 4 mL of 4% SDS/6% trichloroacetic acid and then 3 × 2 mL 6% trichloroacetic acid. After drying, radioactivity bound to the filters was counted in a scintillation counter. Negative controls were also performed without protein substrate or using enzymes that were previously inactivated by incubation for 5 min at 90°C. Each substrate protein was reacted under standard conditions with vehicle, specific transferase inhibitor or ZOL. The level of prenylation was expressed as a percentage of maximum incorporation of [3H]-prenyl for each substrate, as determined by allowing the uninhibited reaction to go to completion.
[14C]-Cholesterol biosynthesis in PC3 cells
The experiment was conducted as described by Awad . Briefly, PC3 cells were seeded in 6-well plates and treated with ZOL for 2, 22, 46 hours and [14C]-mevalolactone (2.25 μCi/ml) was added to the medium for 2 hours more, then the medium was removed, the cells washed twice with PBS, and treated by 1 ml of NaOH (2 M). The mixture was incubated at 37°C for 30 min. and then 0.5 ml of methanol containing 100 μg of non-radioactive cholesterol were added. Saponification took place at 70°C for 1 hour and unsaponified lipids were hexane extracted three times. After drying (under argon) the pellets were dissolved in chloroform and separated by thin layer chromatography (silica gel F/ethyl acetate). [14C]-Cholesterol was revealed and quantified by autoradiography with Phophorimager® (Molecular Dynamics).
Zoledronic acid inhibits proliferation of LNCap, PC-3 and PNT1-A cells
Inhibition of proliferation by ZOL is reversed by geranyl-geraniol
ZOL impairs in vivo geranyl-geranylation of Rap1A without impairing farnesylation of HDJ-2
ZOL impairs Rap1A geranyl-geranylation by preventing GGPP production and not GGPP incorporation into the protein
So, inhibition of geranyl-geranylation by ZOL appears to result from a blockade in GGPP synthesis from FPP rather than from the inhibition of the enzymatic transfer of GGPP onto its protein substrate.
ZOL impairs LPA-induced formation of stress fibers, a cellular effect dependent on RhoA geranyl-geranylation
ZOL Inhibits cholesterol biosynthesis up to 24 hours
Although zoledronic acid is widely used in metastatic prostate cancer management, few data are available about its molecular effects in prostatic cells [15, 20, 21]. Our work shows that, in prostatic cells, ZOL impairs protein geranyl-geranylation but not farnesylation. This result contrasts with the well-documented in vitro inhibition of FPP synthase displayed by many N-BPs [4–6, 33] including ZOL . However, conflicting results about the FPP synthase inhibition by N-BPs have been obtained in vivo. Indeed, some studies support the inhibition of FPP synthase, impairing FPP, and consequently GGPP, production [7–9, 15, 17], whereas other teams suggest the existence of an additional enzymatic target of N-BPs, downstream of FPP synthase [11, 12, 34]. Our results with ZOL lend further support to this latter hypothesis and corroborate two other works performed in mammary MDA-MB-231  and ovarian Caov-3  tumoral cell lines. In the first report , it was shown that the anti-invasive effect of ZOL resulted from an inhibition of RhoA geranyl-geranylation. In alendronate-treated Caov-3 cells, Sawada et al. showed inhibition of Rho activation through GGPP depletion and suggested that this inhibition accounted for the inhibition of LPA-induced stress fiber formation they observed. This biological effect is typically related to RhoA activity.
In the present work it is shown that the main biological activities of ZOL are sustained by its inhibitory effect of protein geranyl-geranylation. We show by study of cholesterol biosynthesis that this impairment of geranyl-geranylation is time-dependently related to an inhibition of FPPsynthase followed by, or concomitant with, inhibition of GGPPsynthase. In vitro studies show that this effect is not related to inhibition of GGTase I
We thanks C. Giamarchi for helpful discussions and technical contributions
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