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CELLULAR AND MOLECULAR
Department of Pharmacology (A.D., R.J.H.), Interdisciplinary Program in Molecular and Cellular Biology (A.J.W., R.J.H.), and Department of Internal Medicine (K.M.L., L.A.V., S.E.D., R.J.H.), University of Iowa, Iowa City, Iowa
Received for publication
September 27, 2007
Accepted
December 13, 2007.
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Abstract |
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). The statins inhibit HMG-CoA reductase (HMGCR), depleting cells of downstream isoprenoids, including farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) (Brown and Goldstein, 1980). FPP is a key intermediate in synthesis of cholesterol, and its depletion results in decreased de novo cholesterol synthesis (Andersen and Dietschy, 1978) leading to increased low-density lipoprotein receptor expression and decreased serum low-density lipoprotein cholesterol in vivo (Goldstein and Brown, 1990). The isoprene moieties from FPP and GGPP are also post-translationally incorporated into several proteins, including many members of the Ras family of small GTPases, which control cell growth and proliferation (Takai et al., 2001), and the Rho family of GTPases, which are important mediators of cell migration (Burridge and Doughman, 2006). Because prenylation is necessary for the activation of small GTPases, statins have been investigated as potential cytotoxic agents for use in cancer chemotherapy (Swanson and Hohl, 2006).
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). These drugs have also been shown in the laboratory to induce apoptosis, and they have direct growth inhibitory effects on malignant cells (Shipman et al., 1997). Like statins, the clinically used NBPs deplete cells of isoprenoid diphosphates, resulting in the inhibition of posttranslational protein prenylation (Reszka and Rodan, 2004). In particular, the NBPs inhibit FPP synthase (FDPS) (van Beek et al., 1999b; Dunford et al., 2001). However, their cellular effects, including induction of apoptosis, may be largely a result of downstream GGPP depletion (Fisher et al., 1999; van Beek et al., 1999a). Based on this rationale, we developed a series of isoprenoid-containing bisphosphonates that specifically inhibit geranylgeranyl diphosphate synthase (GGDPS) (Shull et al., 2006; Maalouf et al., 2007; Wiemer et al., 2007). We have shown that several of these compounds, including digeranyl bisphosphonate (DGBP), can potently inhibit GGDPS in vitro (Wiemer et al., 2007). We have also shown that inhibition of cellular GGDPS leads to depletion of intracellular GGPP levels and not depletion of FPP (Wiemer et al., 2007).
Both statins and NBPs have limited potential as single-agent treatments of malignancies. Inhibition of protein prenylation by the statins does not occur at standard concentrations for treatment of hypercholesterolemia (Lewis et al., 2005). NBPs are limited as anticancer agents because of high bone affinity and low plasma concentrations (Green and Clezardin, 2002). Therefore, if these agents are to be useful for treatment of malignant disease, it is possible that they will need to be used in combination with other agents. This strategy has led to the identification of an increasing number of synergistic interactions between inhibitors of isoprenoid biosynthesis and chemotherapeutic agents (Santini et al., 2006). For example, statins are synergistic with cytosine arabinoside (Holstein and Hohl, 2001a) and paclitaxel (Holstein and Hohl, 2001b). NBPs are synergistic with farnesyl transferase inhibitors (Caraglia et al., 2004), paclitaxel (Jagdev et al., 2001), and imatinib (Kuroda et al., 2003). It is noteworthy that the combination of statins and NBPs is synergistic (Schmidmaier et al., 2006).
Although HMGCR and FDPS have now been extensively studied as therapeutic targets, GGDPS has not. Our recent advances in the design of potent, specific, and cell-permeable GGDPS inhibitors allow the consequences of GGDPS inhibition to be studied. In this study, we show for the first time that inhibition of GGDPS inhibits growth and induces apoptosis through at least two mechanisms, GGPP depletion and more surprisingly, the accumulation of FPP. It is note-worthy that inhibition of GGDPS by digeranyl bisphosphonate is synergistic with either HMG-CoA reductase inhibition by lovastatin or FDPS inhibition by zoledronate.
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Materials and Methods |
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DNA Synthesis Assays. K562 cells were incubated in 96-well plates and treated with compounds as described previously (Shull et al., 2006). After 22 h, 20 µl of [3H]thymidine (0.1385 TBq/mmol; 3.75 Ci/mmol in media) was added to each well. At 24 h, cells were filtered through glass microfiber paper using a Brandel (Gaithersburg, MD) cell harvester. [3H]Thymidine incorporated into cellular DNA was quantified by scintillation counting.
Annexin V and PI Analysis. Annexin V and PI analysis was adapted from the technical data sheet for fluorescein isothiocyanate annexin V (556419; BD Biosciences PharMingen, San Diego, CA), with slight modifications. Treated cells were transferred to 1.5-ml microcentrifuge tubes, they were centrifuged at 1000 rpm for 10 min, and then the supernatant was aspirated. Cells were resuspended in 500 µl of buffer (10 mM HEPES, 150 mM NaCl, 1 mM MgCl2,5mM KCl, and 1.8 mM CaCl2, pH 7.4), and they were transferred to polystyrene test tubes. Five microliters of FITC annexin V was added, and the cells were vortexed and then incubated 15 min at room temperature (RT). Ten microliters of 50 µg/ml PI solution (Sigma-Aldrich, St. Louis, MO) was added to the cell suspension, and the suspension was vortexed. Samples were analyzed using FACS-can (BD Biosciences, Franklin Lakes, NJ).
Western Blot Analysis. Protein concentrations were determined using the bicinchoninic acid method (Pierce Chemical, Rockford, IL). All proteins except poly(ADP-ribose) polymerase (PARP) were resolved by electrophoresis on a 12% gel, and they were transferred to a polyvinylidene difluoride membrane. PARP was resolved using 7.5% gels. Primary and secondary antibodies were added sequentially for 45 min, and proteins were visualized using an enhanced chemiluminescence detection kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Anti pan-Ras was obtained from InterBiotechnology (Tokyo, Japan). Rap1a (sc-1482), Rab6 (sc-310), PARP (sc-7150), and 
-tubulin (sc-8035) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Caspase-3 (9662) was obtained from Cell Signaling Technology Inc. (Danvers, MA). Horseradish peroxidase-conjugated anti-mouse secondary was obtained from GE Healthcare, and Horseradish peroxidase-conjugated anti-goat was obtained from Santa Cruz Biotechnology, Inc. After visualization, proteins were quantified using ImageJ software (http://rsb.info.nih.gov/ij/).
TUNEL Assay. Promega (Madison, WI) TUNEL assay kit (G3250) was performed according to the manufacturer's protocol. Cells were washed twice in PBS, resuspended in 0.5 ml of PBS, and fixed by the addition of 5 ml of methanol-free formaldehyde for 20 min on ice. Cells were washed twice, 5 ml of 70% ice-cold ethanol was added, and cells were stored overnight at –20°C. Washing in PBS was repeated, and cells were resuspended in 1 ml of PBS. The contents were centrifuged, and the cell pellet was resuspended in 80 µl of equilibrium buffer and incubated at RT for 5 min. The contents were centrifuged, and cell pellet was resuspended with 50 µl of recombinant terminal deoxynucleotidyl transferase incubation buffer, incubated at 37°C for 1 h in the dark. Reaction was terminated by addition of 1 ml of 20 mM EDTA. Cells were washed twice in 1 ml of 0.1% Triton X-100 solution in PBS containing 5 mg/ml bovine serum albumin, resuspended in 0.5 ml of 5 µg/ml PI solution containing 250 µg of DNase-free RNase A, and then they were incubated at RT for 30 min, and finally analyzed by flow cytometry.
FPP and GGPP Quantification. FPP and GGPP levels were determined as described previously (Tong et al., 2005). In brief, isoprenoid diphosphates were extracted, and they were used as substrates for incorporation into fluorescent CAAX peptides by farnesyltransferase or geranylgeranyl transferase. Prenylated fluorescent peptides were quantified by fluorescence detection.
Quantification of mRNA. Treated cells were lysed with Invitrogen (Carlsbad, CA) TRIzol reagent, and total RNA were extracted accordingly. An ABI reaction kit (Applied Biosystems, Foster City, CA) was used to synthesize the cDNA for each condition by reverse transcription. Primers for specific genes were mixed with SYBR Green intercalating dye, and they were added to cDNA. PCR was allowed to proceed for 40 cycles. Data were analyzed using ABI SDS 2.3 software (Applied Biosystems), normalized to 18S ribosomal RNA, and quantities were determined using the relative standard curve method as described by the manufacturer. Primers were designed using PrimerQuest (Integrated DNA Technologies, Inc., Coralville, IA). The PCR product was designed to be 200 to 400 base pairs, and primers were checked for specificity with Basic Local Alignment and Search Tool. The following primers were used: HMGCR (5'-ACAGGCTTGAATGAAGCTTTGCCC-3', 5'-GACATGCAGCCAAAGCAGCACATA-3'), FDPS (5'-CTTCCTGCAGAGTTCCTATCAGAC-3',5'-TCTCCAGCAGGATCTTCTTGGCAT-3'), and SQS (5'-ACTTCCCAACGATCTCCCTTGAGT-3',5'-TCCAAACCTCTTGAGGCCAGAACT-3').
Isobologram and Combination Index Analysis. Isobolograms were generated using Calcusyn software (Biosoft, Cambridge, UK). Combination index (CI) values were calculated according to the method of Chou and Talalay (1984) as described in the software manual. For each drug, 48-h IC50 values were determined by thymidine incorporation. Concentration-response curves were generated for each drug and combination using IC50 values as the highest concentration and four 80% dilutions.
Statistical Analysis. Unpaired two-tailed t tests were used to calculate statistical significance. Unless otherwise indicated, comparisons were done relative to the control. All columns in bar graphs represent the mean of the indicated number of replicates. Error bars on graphs represent S.E. An level of 0.05 was set as the level of significance.
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Results |
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Inhibition of Geranylgeranyl Diphosphate Synthase Leads to Induction of the Caspase Cascade in K562 Cells. To confirm the flow cytometric results, which showed that DGBP induces apoptosis, cells were treated with DGBP, and they were assessed for cleavage of PARP and caspase-3 by Western blot analysis. PARP is a DNA repair enzyme that is cleaved during apoptosis by executioner caspases such as activated caspase-3 (Schreiber et al., 2006). Caspases are cysteinyl aspartate-specific proteases that are activated by cleavage during early apoptosis (Thornberry and Lazebnik, 1998). Etoposide (50 µM), which is a well-characterized activator of apoptosis, was used as a positive control. DGBP, like etoposide, induced time-dependent cleavage of PARP as demonstrated by the appearance of an 85-kDa band (Fig. 2C). PARP cleavage was first observed at 24 h, and the effect was more pronounced after 72 h, analogous to what was observed with etoposide. DGBP treatment also leads to the cleavage and activation of caspase-3 as indicated by the appearance of a 17-kDa cleavage product. As expected, etoposide treatment resulted in caspase-3 cleavage. These results indicated that DGBP activates the apoptosis machinery, leading to caspase-3 activation, PARP cleavage, and eventual cell death.
Geranylgeraniol Supplementation Prevents Digeranyl Bisphosphonate-Induced Inhibition of Protein Geranylgeranylation. To determine whether product supplementation could prevent DGBP-mediated inhibition of protein geranylgeranylation, cells were treated with GGOH, which is phosphorylated to form GGPP (Crick et al., 1997). Cells were treated for 48 h with DGBP in the presence or absence of GGOH, and then they were analyzed for inhibition of Rap1a and Rab6 geranylgeranylation by Western blot analysis (Fig. 3). To maximize inhibition of prenylation and to limit apoptotic effects, DGBP was used at a concentration of 40 µM for this experiment. Both Rap1a and Rab6 are exclusively geranylgeranylated; Rap1a is a substrate for geranylgeranyl transferase (GGTase) I, whereas Rab6 is a substrate for GGTase II. It should be noted that the antibody for Rap1a is for the C terminus, and as such, it detects only the unmodified form. The antibodies for Rab6 and Ras detect both modified and unmodified forms.
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Unlike HMG-CoA Reductase Inhibition, Apoptosis Induced by Geranylgeranyl Diphosphate Synthase Inhibition Is Not Fully Reversed by Product Supplementation. To determine whether product supplementation could prevent the apoptosis induced by inhibition of GGDPS as indicated by PARP and caspase-3 cleavage, cells were treated with 100 µM DGBP for 72 h (Fig. 4A). As controls, cells were treated with either lovastatin, zoledronate, or GGTI-298, a peptidomimetic inhibitor of geranylgeranyl transferase I (Lerner et al., 1995). We were surprised to find that the addition of 10 µM exogenous GGOH did not prevent the ability of 100 µM DGBP to induce apoptotic effects (Fig. 4A, lanes 5 and 9). In fact, addition of 10 µM GGOH to 100 µM DGBP enhanced the cleavage of both PARP and caspase-3, whereas GGOH had no effects on PARP and caspase-3 on its own (Fig. 4A, lane 7). This contrasts to 50 µM lovastatin-induced PARP and caspase-3 cleavage, which was completely prevented by the addition of 1 mM mevalonate (Fig. 4A, lanes 3 and 8). Treatment with 250 µM zoledronate (Fig. 4A, lane 4) displayed minor amounts of cleavage, whereas 20 µM GGTI-298 did not induce any PARP and caspase-3 cleavage (Fig. 4A, lane 6). We performed a TUNEL assay to detect DNA fragmentation that results from the apoptotic signaling cascade, using the same conditions (Fig. 4B). As with PARP and caspase-3 cleavage, 50 µM lovastatin induced DNA fragmentation (Fig. 4B, column 3) that was prevented by the addition of 1 mM mevalonate (Fig. 4B, column 8), whereas 10 µM GGOH (Fig. 4B, column 9) was not able to prevent DNA fragmentation resulting from 100 µM DGBP (Fig. 4B, column 5). GGOH (10 µM) (Fig. 4B, column 7) and 20 µM GGTI (Fig. 4B, column 6) did not lead to DNA fragmentation. Zoledronate (250 µM) (Fig. 4B, column 4) lead to DNA fragmentation but not as well as DGBP or lovastatin. Mevalonate alone had no effect on cleavage of PARP or caspase-3 and DNA fragmentation (data not shown).
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Inhibition of Geranylgeranyl Diphosphate Synthase Leads to Apoptosis through Two Different Mechanisms. To further explore the effects seen by combinations of DGBP and GGOH, cells were treated with low and high concentrations of DGBP, and levels of intracellular FPP and GGPP were measured (Fig. 5). Treatment with DGBP concentration-dependently decreased GGPP, whereas it increased FPP. GGOH partially reversed both the increase in FPP and decrease in GGPP.
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). Isoprenoid alcohols, but not diphosphates, inhibit choline phosphotransferase leading to depletion of phosphatidylcholine (PC) (Voziyan et al., 1993; Miquel et al., 1998). Cells were treated with low (25 µM) or high (100 µM) DGBP in the presence or absence of GGOH (10 µM) or PC (100 µM) (Fig. 6). GGOH (10 µM) prevented apoptotic effects caused by low (25 µM) DGBP (Fig. 6, A and B). Although 10 µM GGOH completely prevented staining, 100 µM PC was not able to significantly impair, annexin V and PI staining (Fig. 6A, columns 4–6) and PARP cleavage (Fig. 6B, lanes 4–6) caused by 25 µM DGBP. However, PC was able to partially impair apoptotic effects caused by high (100 µM) DGBP (Fig. 6, A and C). Addition of 100 µM PC prevented some annexin V and PI staining (Fig. 6A, columns 7 and 9) and most PARP cleavage (Fig. 6C, lanes 4 and 6) caused by 100 µM DGBP. In contrast, the addition of 10 µM GGOH to 100 µM DGBP lead to enhanced annexin V and PI staining (Fig. 6A, columns 7 and 8) and PARP cleavage (Fig. 6C, lanes 4 and 5). Etoposide-induced apoptotic effects were not significantly altered by the addition of PC (Fig. 6, A–C, lanes/columns 2 and 3). Annexin V and PI staining and PARP cleavage were not affected by 10 µM GGOH or 100 µM PC (data not shown).
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Inhibition of Geranylgeranyl Diphosphate Synthase Alters Steady-State mRNA Levels of Isoprenoid Biosynthetic Enzymes. Because some of the effects of DGBP seemed to result from accumulation of FPP, whose downstream sterol products are known to regulate the isoprenoid biosynthetic machinery through transcriptional events (Brown and Goldstein, 1997), we hypothesized treatment with DGBP would result in decreased mRNA expression of HMGCR, FDPS, and SQS. Cells were treated with DGBP, and then they were tested for altered expression of enzymes involved in isoprenoid biosynthesis via quantitative real-time PCR (Table 1). As characterized previously, treatment with either lovastatin or zaragozic acid resulted in increased mRNA expression of HMGCR, FDPS, and SQS. Supporting our hypothesis, treatment with DGBP resulted in decreased expression of HMGCR, FDPS, and SQS mRNA.
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Digeranyl Bisphosphonate-Induced Inhibition of Geranylgeranyl Diphosphate Synthase Is Synergistic with Lovastatin and Zoledronate. To further examine the consequences of GGDPS inhibition in the context of other protein geranylgeranylation inhibitors, cells were tested for synergistic interactions between DGBP and lovastatin, zoledronate, or GGTI-298. For these studies, concentration-response curves for each compound were constructed using [3H]thymidine incorporation at the 48-h time point. All drugs were tested alone or in combination with constant ratios of DGBP according to the method of Chou and Talalay (1984). The data were analyzed in two ways. First, isobologram analyses were conducted (Fig. 7). A general isobologram with a line of additivity and regions of synergy and antagonism is shown (Fig. 7A). Lovastatin and DGBP were strongly synergistic (CI50 = 0.03) for inhibition of K562 cell growth (Fig. 7B). Zoledronate was also synergistic with DGBP (CI50 = 0.84) (Fig. 7C), but not to the extent of the synergy with lovastatin. GGTI-298 was antagonistic to DGBP (CI50 = 1.64) (Fig. 7D). Although isobologram analysis can determine whether a combination of compounds is synergistic and approximate the magnitude of the synergistic interaction, it does not quantify this at the experimental concentrations per se. Therefore, the CI values for each experimental combination of DGBP with lovastatin or zoledronate were calculated (Table 2). At all of the experimental concentrations tested, lovastatin and DGBP displayed stronger synergy than the combination of zoledronate and lovastatin (CI50 = 0.64). This result has been observed in RPMI-8226 and U937 cells as well (data not shown).
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To further support the synergy observed in [3H]thymidine incorporation assays, CI analysis was performed after annexin V and PI staining on cells treated with combinations of lovastatin and DGBP (Table 3). Similar to the [3H]thymidine incorporation assays, the annexin V and PI staining assays revealed a synergistic interaction toward total cell death (CI50 = 0.01). More specifically, these assays also demonstrated a synergistic interaction occurs both for cells in early (CI50 = 0.76) and late apoptosis (CI50 = 0.50).
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Digeranyl Bisphosphonate Potentiates Lovastatin-Induced Inhibition of Protein Geranylgeranylation. Because both lovastatin and DGBP inhibit protein geranylgeranylation through different but overlapping mechanisms, combinations of the two compounds were tested for the ability to inhibit protein geranylgeranylation (Fig. 8). Cells were treated with lovastatin and DGBP alone or in combination. To observe synergistic effects, it was necessary to use lower concentrations of lovastatin and DGBP. Both lovastatin and DGBP exhibited submaximal inhibition of geranylgeranylation at concentrations below 10 and 20 µM, respectively. Concentrations of 1.1 µM lovastatin and 2.2 µM DGBP had minimal effects, whereas the combination of these concentrations was sufficient to impair geranylgeranylation (Fig. 8, lanes 3, 7, and 11). This effect was also pronounced at concentrations of 3.3 µM lovastatin and 6.6 µM DGBP (Fig. 8, lanes 4, 8, and 12). It is noteworthy that the combination of lovastatin and DGBP had an antagonistic effect on inhibition of Ras farnesylation. Although lovastatin caused the accumulation of an unmodified upper Ras band (Fig. 8, lanes 4 and 5), DGBP did not (Fig. 8, lanes 8 and 9). When combined, DGBP prevented the inhibition of Ras farnesylation induced by lovastatin (Fig. 8, lanes 12 and 13). Quantification of the Rap1a bands and Rab6 upper bands further supports the synergistic interaction of lovastatin and DGBP toward geranylgeranylation inhibition.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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), FDPS (Shipman et al., 1997), and protein prenyltransferases (Lebowitz et al., 1997), inhibition of GGDPS may be a viable therapeutic strategy. Inhibition of GGDPS has two consequences, depletion of cellular GGPP and an accumulation of FPP. At any concentration of DGBP, the inhibition of protein geranylgeranylation can be prevented by addition of exogenous GGOH. At low amounts of GGDPS inhibition, apoptotic consequences are able to be prevented by exogenous GGOH. This agrees with previous studies that showed GGOH reverses apoptosis induced by the clinical NBPs (van Beek et al., 1999a). At greater GGDPS inhibition, apoptotic effects are not prevented by exogenous GGOH, rather they are enhanced. It is possible that increased cellular FPP (and FOH) levels caused by GGDPS inhibition in the presence of exogenous GGOH are high enough to inhibit cellular cholinephosphotransferase (Miquel et al., 1998). Our data support this conclusion because apoptosis induced by high concentrations of DGBP can be partially prevented by addition of exogenous PC. It is possible that PC cannot fully prevent apoptosis because it does not prevent the inhibition of protein geranylgeranylation caused by GGDPS inhibition.
In addition to inhibition of PC synthesis, there are at least three other well characterized consequences of FPP or FOH accumulation that are relevant to regulation of isoprenoid biosynthesis. First, FPP is converted to squalene and eventually cholesterol, which when elevated decreases transcription of the enzymes responsible for its synthesis (Brown and Goldstein, 1997). Our results clearly show decreased mRNA levels of three of these enzymes, HMGCR, FDPS, and SQS associated with FPP accumulation caused by GGDPS inhibition. The fact that these changes were seen in media containing serum is consistent with the requirement of endogenous sterol synthesis to regulate gene expression. However, further studies of GGDPS in serum-free media may reveal more dramatic changes. Second, FPP and FOH have been shown conclusively to increase HMGCR degradation in pulse-chase experiments (Meigs and Simoni, 1997). Third, both FPP and FOH have been shown to inhibit the mevalonate kinase reaction (Dorsey and Porter, 1968).
We have also shown for that first time that inhibition of GGDPS in combination with inhibition of either HMGCR or FDPS results in synergistic inhibition of cell growth and induction of apoptosis. It is possible that both the FPP accumulation and GGPP depletion caused by GGDPS inhibition serve to enhance lovastatin-induced apoptosis. In contrast, lovastatin and zoledronate both similarly deplete FPP and GGPP and lack the apoptotic effects caused by FPP accumulation. This line of reasoning also explains why the combination of zoledronate and DGBP is more synergistic than the combination of zoledronate and lovastatin.
The data presented herein suggest that inhibition of GGDPS may be a good therapeutic strategy either alone on in combination with other isoprenoid pathway inhibitors. DGBP may be useful alone or in combination with statins for inhibition of cancerous cell growth or other cellular processes that are dependent upon geranylgeranylation. Ongoing studies will further evaluate effects of GGDPS inhibition on cancer and bone disorders.
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Acknowledgements |
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Footnotes |
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A.D. and A.J.W. contributed equally to this article.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: HMGCR, HMG-CoA reductase; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; NBP, nitrogenous bisphosphonate; FDPS, farnesyl diphosphate synthase; GGDPS, geranylgeranyl diphosphate synthase; DGBP, digeranyl bisphosphonate; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; PI, propidium iodide; PCR, polymerase chain reaction; PARP, poly(ADP-ribose) polymerase; RT, room temperature; PBS, phosphate-buffered saline; CI, combination index; GGOH, geranylgeraniol; GGTase, geranylgeranyl transferase; FOH, farnesol; PC, phosphatidylcholine; SQS, squalene synthase; Lov, lovastatin; GGTI-298, N-4-[2(R)-amino-3-mercaptopropyl]amino-2-naphthylbenzoyl-(L)-leucine methyl ester trifluoroacetate.
Address correspondence to: Dr. Raymond J. Hohl, University of Iowa, SE 313 GH, Iowa City, IA 52242. E-mail: raymond-hohl{at}uiowa.edu
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