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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Mol Cancer Ther. Author manuscript; available in PMC 2010 November 1.
Published in final edited form as:
PMCID: PMC2888841
NIHMSID: NIHMS113203

In vivo antitumor effect of a novel inhibitor of protein geranylgeranyltransferase I

Abstract

Protein geranylgeranyltransferase-I catalyzes protein geranylgeranylation which is critical for the function of proteins such as Rho, Rac and Ral. We previously identified several small molecule inhibitors of GGTase-I from an allenoate-derived compound library and showed that these compounds exhibit specific inhibition of GGTase-I resulting in the inhibition of proliferation associated with the induction of G1 cell cycle arrest of a variety of cancer cell lines. Since inhibition of GGTase-I is expected to suppress tumor growth, we investigated in vivo effects of one of these GGTIs, P61A6, by using a human pancreatic cancer xenograft model in mice. The new compound GGTI P61A6 showed an excellent antitumor effect. Intraperitoneal administration of P61A6 significantly suppressed tumor growth of the PANC-1 xenograft. Even once per week administration of GGTI was enough to suppress tumor growth. Immunohistochemical examination indicated the inhibition of cell proliferation in the tumors by P61A6 treatment, but neither apoptosis nor antiangiogenesis was observed. Increased cytosolic localization of proteins such as Rap1 and RhoA in tumors was observed. In addition, the enzyme activity of GGTase-I in tumors was inhibited. Pharmacokinetic analysis showed that the plasma half-life of GGTI is almost 6 hours, suggesting its prolonged effect. These data suggest that the novel GGTI compound P61A6 is an excellent chemotherapeutic drug candidate for human pancreatic cancer. They also provide evidence that protein geranylgeranyltransferase-I may be a valid target for cancer therapy.

Keywords: GGTI, xenograft, pancreatic cancer, geranylgeranyltransferase I

Introduction

Recent studies on protein geranylgeranylation point to the significance of this posttranslational modification in oncogenesis. Proteins such as RhoA, RhoC, Rap1 and Ral are geranylgeranylated (1). Discovery of Dlc1, Rho-GAP, as a major tumor suppressor suggests that activation of the Rho type proteins is wide spread in cancer (2, 3). RhoC is a key protein that is required for tumor metastasis (4, 5). Ral proteins are activated in more than 90% of pancreatic duct adenocarcinoma cases (6). Protein geranylgeranylation is catalyzed by protein geranylgeranyltransferase I (GGTase-I), an enzyme that adds a C20 geranylgeranyl group to the cysteine of proteins with the carboxy-terminal tetrapeptide consensus sequence CAAL (C is cysteine, A is any aliphatic amino acid, and the C-terminal residue is leucine or phenylalanine) (711). Characterization of mice with conditional knockout of GGTase-I showed that the GGTase-I deficiency results in reduced oncogenic K-ras-induced lung tumor formation and dramatically increased survival (12). GGTase-I deficient cells showed proliferation inhibition and accumulation of p21CIP1/WAF1, pointing to the importance of GGTase-I in cell proliferation and cell cycle progression (12).

These observations prompted us and others to design GGTase-I inhibitors (GGTIs) as potential anticancer drugs (1319). To date, several GGTI compounds have been developed, such as GGTI-298 (20), GGTI-2154 (15), GGTI-2166 (21) and GGTI-286 (22). While these are derived from CAAL peptide, there are also non-peptidomimetic inhibitors. GGTI-DU45 was identified via high-throughput screening of a compound library(16). We have recently established a new chemical compound library of more than 4000 allenoate-derived compounds, screened them for inhibitors of human GGTase-I and identified a number of GGTIs which can be divided into two groups: one group containing a tetrahydropyridine ring as its core scaffold and the other group having a dihydropyrrole ring as its core scaffold (18, 23). These GGTI compounds inhibit the protein modification and block membrane association and function of Ral, Rho and Rap subfamilies, cause cell cycle arrest at the G1 phase, and suppress the growth of several human cancer cell lines including leukemic, pancreatic cancer, and breast cancer (18).

Anti-tumor efficacy of GGTI compounds has been reported. Demonstration of the antitumor efficacy of GGTI-2154 was made using the human lung adenocarcinoma A549 xenograft model (24). By using the MMTV-ν-Ha-Ras model, Sun et al (15) showed that GGTI-2154 induced apoptosis, tumor regression, and differentiation as well as inhibited oncogenic and tumor survival pathways in nude mice. GGTI-2 was used by Lobell et al. to inhibit growth of tumor xenografts in mice in combination with FTI (25). In this experiment, administration of 3 mg/kg per day of GGTI-2 alone was not deleterious; however, administration of 30 – 100 mg/kg per day caused cellular depletion in the bone marrow and spleen. These observations suggest that low concentrations of GGTIs are well tolerated. The challenge will be to achieve significant inhibition of geranylgeranylation at low concentrations of GGTI so that any deleterious effects will be minimal.

In this paper, we have reassessed the issue of using GGTIs to inhibit tumor growth in mice by using our novel GGTI compounds. We show that GGTI P61A6 has an excellent antitumor effect in the human pancreatic cancer xenograft model in SCID mice. Intraperitoneal administration of GGTI P61A6 at 1.16 mg/kg/day caused a significant suppression of the growth of the human pancreatic cancer xenograft in mice. Even once per week administration of GGTI was enough to cause a similar level of suppression, and dose dependent efficacy was observed. Immunohistochemical examinations indicated the inhibition of cell proliferation in the tumors by P61A6 treatment, but neither apoptosis nor antiangiogenesis was observed. In addition, inhibition of geranylgeranylation in tumors was observed. These data suggest that the novel GGTI compound P61A6 is an excellent chemotherapeutic drug candidate for human pancreatic cancers.

Materials and Methods

GGTI compounds

The allenoate-derived compound library was synthesized as described in our previous publication (18, 23). A 20 mM stock solution of GGTI P61A6 (Fig. 1A) in DMSO was kept at −20°C until use.

Figure 1
A. The molecular structure of GGTI P61A6. B. Antitumor Effect of GGTI P61A6. Treating with 1.16 mg/kg P61A6 (3 times weekly, every other day) in treated mice (--[box]--) or 0.2 ml 0.9% NaCl in control mice (-■-) was started from 14th day after ...

Cells and Culture

The human pancreatic cancer cell line, PANC-1, obtained from American Type Culture Collection, was maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal calf serum (Sigma, MO), 2% L-glutamine, 1% penicillin and 1% streptomycin. The medium was routinely changed every 3 days, and the cells were separated by trypsinization before reaching confluency.

Tumorigenesis in SCID Mice

The stock solution of GGTI P61A6 was diluted in saline to give a final concentration of 160 µM immediately before administration. Female 5-week-old C.B-17 SCID mice were obtained from Charles River Laboratories. 5×106 of PANC-1 cells in 0.2 ml DMEM were subcutaneously injected into the right lateral abdominal wall of the mice. Two weeks later, mice bearing tumors around 3 mm in diameter were randomly divided into treatment or vehicle groups in each experiment. 0.25 ml of different concentrations of GGTI P61A6 solution (160 µM or 1.16 mg/kg of body weight, or 80 µM or 0.58 mg/kg of body weight, or 40 µM or 0.29 mg/kg of body weight, or vehicle (DMSO in saline, DMSO final concentration=0.8%)) was injected intraperitoneally from the right lower corner of the abdomen into the mice at different injection schedules, until the end of the experiment. Tumor volume and body weight were measured every other day, and tumor volume was calculated using the following formula: tumor volume = (4/3) × 3.14 × (L/2×W/2×W/2), where L is the length of the tumor and W is its width. On the second and 10th day of the experiment, and before sacrifice, 150 µl of blood was collected from retroorbit for hematological and serum biochemical examinations.

In vivo Pharmacokinetic Evaluation

For pharmacokinetic evaluation, 3 female mice, 5 weeks old, were intraperitoneally injected with GGTI P61A6 (1.16 mg/kg of body weight). Blood samples (60 µL) were drawn from retro-orbital at 15 min, 30 min, 60 min, 2 hr, 4 hr, and 24 hr from each mouse, collected in EDTA treated tubes, mixed well, and frozen immediately. Plasma was harvested and kept at −20 °C until assayed.

Concentrations of P61A6 in plasma were determined using a validated analytical procedure based on high-performance liquid chromatography. LC-MS/MS analyses were carried out using a SCIEX API3000 triple quadrupole mass spectrometer (PE Sciex Instruments, Boston, MA) operating in electrospray ionization mode. Chromatography was carried out using gradient elution (water-acetonitrile) on a Kromisil C18 reverse-phase column at a flow rate of 1 mL/min. Plasma compound concentrations were determined using a 7 point calibration curve derived from peak areas obtained from serially-diluted solutions of P61A6. Pharmacokinetic (PK) analysis was performed by noncompartmental analysis using PK Solutions (version 2.0) software (Summit Research Services).

Effects of GGTI P61A6 on prenylation of RhoA and Rap1 in mouse tumor samples

In their prenylated forms, proteins such as RhoA and Rap1 associate with cellular membranes (26). Inhibition of geranylgeranylation dissociates these proteins from the membrane. Therefore, to test for geranylgeranylation inhibition, membrane and cytosolic fractions were prepared by homogenizing tumor biopsies in ice-cold hypotonic buffer [10 mM Tris (pH 7.5), 5 mM Mgcl2, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 25 µg/ml leupeptin] following the published protocol (15). The homogenates were then filtered through one layer of nylon mesh and centrifuged at 12,000 × g, and then the supernatant was centrifuged at 100,000 × g for 30 min at 4°C to separate membrane and cytosolic fractions. The membrane fractions were lysed by HEPES lysis buffer [30 mM HEPES (pH 7.5), 1% Triton X-100, 10% glycerol, 10 mM NaCl, 5 mM MgCl2, 25 mM NaF, 1 mM EGTA, 2 mM Na2VO4, 10 µg/ml soybean trypsin inhibitor, 25 µg/ml leupeptin, 10 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, and 6.4 mg/ml 2-nitrophenylphosphate]. The lysates were then separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antibodies against Rap1 (121) or RhoA (26C4) (Santa Cruz Biotechnology, CA).

Effects of GGTI P61A6 on GGTase-I Activity in Tumor

GGTase-I enzymatic activity was determined in collected subcutaneous tumors from mice either treated with GGTI P61A6 or saline solution. Since GGTase-I is a cytosolic enzyme, postmicrosomal fractions (60,000 × g supernatants) were first isolated according to the published protocol (15). Briefly, collected tumors were homogenized in ice-cold homogenizing buffer [50 mM Tris (pH 7.5), 1 mM EGTA, 1 mM DTT, 2 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin, and 10 µg/ml aprotinin], sonicated, and centrifuged at 12,000 × g and at 60,000 × g. The resulting supernatants were used to determine GGTase-I enzymatic activity by following the incorporation of radiolabeled isoprenoid [3H]geranylgeranyl into substrate proteins as described previously (18). Briefly, various amounts of supernatants were used to initiate reactions containing 0.5 µM [3H]GGPP and 2 µM maltose-binding protein-tagged RhoA in reaction buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 µM ZnCl2, 0.01% Triton X-100, and 1 mM dithiothreitol. Reactions were carried out for 15 min at 30°C and spotted onto filter paper and treated with 10% trichloroacetic acid, followed by ethanol and acetone washing. The filter was counted using a scintillation counter.

Immunohistochemistry

The effects of GGTI P61A6 on angiogenesis in the tumor xenograft experiments were investigated by immunohistochemistry. The purified rat anti-mouse CD31 (PECAM-1) monoclonal antibody (BD Biosciences) was used to detect tumor vasculature. Briefly, the subcutaneous tumors were removed at the end of the in vivo experiments, embedded in O.C.T compound (Tissue-Tek), frozen, and microdissected into 4 µm-thick serial sections with a Tissue-Tek Cryo. The tissue sections were fixed with 100% acetone and Carnoys fixative (60% absolute ethanol, 30% chloroform and 10% glacial acetic acid) for 15 min and immersed in methanol containing 0.3% hydrogen peroxide to block endogenous peroxide activity. After incubation with a blocking buffer (2% BSA in PBS buffer), the sections were exposed to the first antibody (1% dilution) for 1 h at room temperature. Biotinylated polyclonal anti-rat Igs (BD Biosciences) was used as the secondary antibody. Peroxide staining was performed for 2–5 min with a solution of 3,3'-diaminobenzidine tetra hydrochloride (DAB) in 50 mM Tris-HCl (pH 7.5) containing 0.001% hydrogen peroxide, and the sections were counterstained with 0.1% hematoxylin. Intratumoral microvessel density (MVD) was analyzed with a KS300 imaging system (Carl Zeiss Vision GmbH). Photographs of the entire area of slides were taken after staining, and all of the vessels were counted on each slide. Microvessel density was calculated as the total number of vessels on each slide/the gross area of the slide.

The TUNEL assay was used to detect apoptosis in the xenograft. Briefly, deparaffinized sections were pretreated with proteinase K and rinsed in PBS-Tween buffer. After blocking in 3% H2O2 in PBS for 10 minutes and rinsing, the sections were incubated in TdT Reaction Buffer for 10 minutes, and then in TdT Reaction Mixture for 2 hours at 37°C. After stopping and rinsing, Streptavidin-HRP in PBS was added and the sections were incubated for 10 min, and then incubated with DAB until the brown staining was obvious. Sections were counterstained with Gill's hematoxylin and dehydrated with ethanol. The images were analyzed with an Olympus microscope/Metamorph and Ariol systems.

DNA synthesis was detected by 5-bromo-2’-deoxyuridine (BrdU) incorporation. BrdU (100 µg/g bodyweight; Sigma-Aldrich Co., St. Louis, MO) was injected into the mice 1 hour before sacrifice. The tumor sections were stained by immunohistochemistry as described above. For quantitation, the tumor slides were scanned and the number of BrdU-positive nuclei was counted and compared with the total number of nuclei in the same section, determined by hematoxylin staining.

Statistical Analysis

All results are expressed as mean±SD. Statistical comparisons were made using Student’s t test after analysis of variance. The results were considered to be significantly different at P value<0.05.

Results

Antitumor Effect of GGTI P61A6

The compound P61A6 (Fig. 1A) was shown to inhibit proliferation of various human cancer cells in our previous study (18). In this study, the compound’s in vivo anti-tumor effect was assessed using mouse xenografts of human cancer cells. To determine the maximum tolerated dose (MTD) in mice, 6 female mice, 5 weeks old, were injected with various doses of P61A6 (Dose range: 0–4.64 mg/kg/day) by intraperitoneal administration once per day for 10 days. Body weight change, visible and/or palpable dermal infection, presence of ascites, and grooming or impaired mobility were closely monitored every day. Also, the body condition scoring system (BCS) was used to evaluate the nutrition status. On the 2nd and 10th day, about 60 µl of blood was collected from retroorbit for hematological and serological examination. On the 10th day, all mice were sacrificed and all organs were collected for histopathological examination. The results show that 10 day treatment with different dosages of P61A6 did not cause any body weight loss, mobility impairment, or histopathological alteration in any mouse but two (Table 1). The two mice showing slight effect were those treated with the two highest doses (4.64 mg/kg and 2.32 mg/kg). These mice showed a slight increase of serum Alanine Aminotransferase (ALT) (540 and 546 U/L on the 2nd and 10th day, respectively, for mouse treated with 2.32 mg/kg; 272 and 267 U/L on the 2nd and 10th day, respectively, for mouse treated with 4.64 mg/kg, normal range 7–227 U/L) and Aspartate Aminotransferase (AST) (345 and 356 U/L on the 2nd and 10th day, respectively for mouse treated with 2.32 mg/kg; 329 and 332 U/L on the 2nd and 10th day, respectively, for mouse treated with 4.64 mg/kg, normal range 37–329 U/L)(See Supplementary Information table S1). Therefore, we decided that 1.16 mg/kg (0.25 ml of 160 µM GGTI in 0.9% NaCl) would be the highest concentration (≈ 20 µM in plasma) to be used for the following xenograft experiments.

Table 1
Determining the maximum tolerance dosage (MTD) of GGTI P61A6.

In the first in vivo experiment, 14 female SCID mice were used. 5×106 PANC-1 cells were subcutaneously implanted into the back of each mouse. After 2 weeks, the mice with tumors around 3 mm in diameter were randomly divided into treatment and control groups (7 mice for each group), and administration of 1.16 mg/kg P61A6 (0.25 ml of 160 µM GGTI in 0.9% NaCl) (3 times per week, every other day) was initiated as described in Materials and Methods. As shown in and 1C, after 33 days of intraperitoneal administration of P61A6, both tumor volumes and tumor wet weight in the treated group was significantly suppressed to approximately 35% and 39% of that in the control mice respectively. Examinations of hematology, serology and tissue pathological alteration did not show any significant changes (data not shown).

The second animal experiment was carried out with 30 female mice (Fig. 2). Similar to the first experiment, the subcutaneous tumors were established on the back of each mouse. Two weeks later, the mice were randomly divided into 5 groups (1 group for control, 4 groups for treated mice, 6 mice for each group). The 6 mice in group 1 were treated with 0.25 ml of 0.9% NaCl every day by intraperitoneal administration, while group 2 mice were treated with 1.16 mg/kg of GGTI P61A6 (0.25ml of 160 µM GGTI in 0.9% NaCl) 6 times per week, group 3 with 1.16 mg/kg of P61A6 (0.25ml of 160 µM GGTI in 0.9% NaCl) once per week, group 4 with 0.58mg/kg of P61A6 (0.25ml of 80 µM GGTI in 0.9% NaCl) 3 times per week (every other day), and group 5 with 0.29 mg/kg of P61A6 (0.25 ml of 40 µM GGTI in 0.9% NaCl) 3 times per week for 44 days. At the end of the experiment, the tumors in control mice grew up to 776 mm3 in average volume. The results on tumor volume are presented in Figure 2A. In the treated groups, inhibition of tumor growth was evident. Administration of GGTI P61A6 in group 2 (1.16 mg/kg of P61A6 6 times per week) significantly suppressed the tumors to approximately 32% of control (Fig. 2Aa). Even treatment with P61A6 once per week resulted in the inhibition of tumor growth to 47% of control mice (Fig. 2Aa). In Figure 2Ab, results of using lower dosages of P61A6 are shown. Treatment with 0.58 mg/kg of P61A6 3 times per week (group 4) effectively suppressed the tumor growth to 62% of the control. However, decreasing the dosage of P61A6 further (0.29 mg/kg, 3 times per week) (group 5) resulted in a diminished tumor-suppressing effect, although transient suppression in the first two weeks was observed (Fig. 2Ab). These results clearly demonstrated the dose-dependent tumor-suppressing effect of GGTI P61A6 in animals (Fig. 2Ac).

Figure 2
Antitumor Effect of GGTI P61A6 with different dosages and frequencies. A: a. Mice with tumors were treated from 14th day with 1.16 mg/kg P61A6 through intraperitoneal with different intervals. Group 1 (-■-): control group, 0.25 ml 0.9% NaCl 6 ...

We did not observe significant body weight loss in treated mice (Fig. 2Ad). To investigate possible toxicity of P61A6, the mice’s organs, such as esophagi, stomach, lung, liver, kidney and spleen were taken when the mice were sacrificed and subjected to hematoxylin and eosin staining. The blood was also collected from the heart for hematology and serology examination. No significant abnormalities were observed (data not shown).

Pharmacokinetics of P61A6

Pharmacokinetic parameters of GGTI P61A6 were determined in mice dosed intraperitoneally at 1.16 mg/kg. As can be seen in Figure 3 and supplementary table S2, P61A6 demonstrated good pharmacokinetic parameters that are amenable to the efficacious dosage amounts. P61A6 had low plasma clearance (CL = 22.7 mL min−1 kg−1) and a relatively small volume of distribution (Vd = 183 mL/kg) in mice, resulting in a terminal half-life of 5.6 h. The time of maximum concentration (Tmax) in plasma occurred at 30 min after dosing with the mean maximal concentration (Cmax) in plasma via intraperitoneal administration being 7.1 µg/mL (Fig. 3 and supplementary table S2).

Figure 3
Plasma concentrations of GGTI P61A6 in mice after intraperitoneal administration. Mice were intraperitoneally injected with GGTI P61A6 (1.16 mg/kg of body weight). Blood (60 µL) were drawn from suborbital at 15 min, 30 min, 60 min, 2 h, 4 h, and ...

P61A6 suppresses cell proliferation in tumors

The growth of transplanted tumors depends significantly on the angiogenic ability of the tumor (27), and inhibition of protein geranylgeranylation and RhoA/RhoA kinase pathway was reported to interfere with angiogenesis (28, 29). Therefore, we first examined the effect of GGTI P61A6 on tumor angiogenesis by immunohistochemical staining using the anti-CD31 antibody in tumors. Microvessel density was counted as described in Materials and Methods. No differences in microvessel density were observed between the tumors in the treatment and control groups (MVD: Control 192±34.5 vessles/mm2; Treated: 200.4±39.4 vessles/mm2) (Fig 4).

Figure 4
Representative immunohistochemistry images of tumors from group 1 and group 2. Mice with tumors were treated from 14th day with 1.16 mg/kg P61A6 through intraperitoneal with 0.25 ml 0.9% NaCl, 6 days per week (group 1) or 1.16 mg/kg of P61A6, 6 times ...

To assess P61A6-mediated apoptosis in xenografts, we examined cells with the TUNEL assay that detects DNA fragmentation characteristic of apoptotic cells in the tumors from treated and control groups. The tumor sections were analyzed with an Olympus microscope/Metamorph and Ariol systems. We did not observe statistically significant differences of the TUNEL positive cells (apoptotic cells) between the mice from the control and treated groups; although larger tumors in control mice seemed to have more apoptotic cells (Fig. 4).

In our previous in vitro study, we found that GGTI P61A6 inhibited the proliferation of various human cancer cell lines and caused G1 cell cycle arrest (18). Therefore, the rate of proliferating cells in the xenograft tumors was also examined by measuring incorporation of bromodeoxyuridine (BrdU) in newly synthesized DNA of replicating cells. Mice were pulsed with a single BrdU injection, killed 1 hour later, and analyzed for BrdU incorporation. These results are shown in Figure 4. Interestingly, the average percentage of BrdU positive cells (S-phase cells) in the P61A6 treated mice tumors is 13.6±4.7%, compared to 20.4±3.4% in the control mice (Fig. 4), suggesting significant inhibition of DNA synthesis of tumor cells by GGTI P61A6. These results suggest that the main effect of GGTI is to inhibit proliferation of tumor cells.

GGTI P61A6 Inhibits GGTase-I activity in pancreatic cancer xenografts in mice

Since proteins such as RhoA and Rap1 are modified by GGTase-I and the prenylation is required for these proteins to be associated with cellular membranes, we assessed whether GGTI treatment inhibits membrane association of these proteins. Membrane and cytosolic fractions were prepared from these tumors by ultracentrifugation and processed for SDS-PAGE Western immunoblotting. The membrane marker, Lactate dehydrogenase (LDH), was used to confirm the separation of cytosol fractions. As shown in Fig. 5A, in tumors from control mice, RhoA and Rap1 proteins were predominantly present in the membrane fractions. Treatment with GGTI P61A6 resulted in a dramatic increase of Rap1 and RhoA in the cytosolic fractions while their association with the membrane fractions decreased.

Figure 5
A. GGTI P61A6 inhibits protein geranylgeranylation in tumors. Inhibition of protein geranylgeranylation was evaluated by determining the proportion of these proteins associated with cellular membrane fractions and cytosolic fractions. Mice with tumors ...

Next, we measured GGTase-I enzymatic activities in tumors from the control and treated groups. GGTase-I activity was examined using the cytosolic fraction of the tumor cells. As shown in Fig. 5B, incorporation of radiolabeled isoprenoid [3H]geranylgeranyl into substrate protein RhoA was significantly inhibited in the extracts of tumors from the treated mice compared to that of the control mice, suggesting significantly reduced GGTase-I enzyme activity. These results suggest that GGTI P61A6 reached the tumors and inhibited GGTase-I in animals.

Taken together, these results demonstrate that the treatment with GGTI P61A6 exhibits a clear and significant inhibition of protein geranylgeranylation in animal xenografts.

Discussion

In the present study, we tested in vivo anticancer effects of a novel GGTI P61A6 using human pancreatic cancer xenograft in SCID mice. GGTI P61A6 markedly inhibited tumor formation to approximately 35% of the control mice when administered 3 times per week at a 1.16 mg/kg concentration. Even treatment with P61A6 once per week inhibited the tumor growth to 47% of control. Moreover, half of the dosage also showed good tumor inhibition in mice. Further decreased dosage failed to inhibit tumors, although a transient suppression after 4 weeks was observed. Thus, P61A6 exhibits excellent anti-tumor activity. The inhibition of tumor growth by treatment once per week is encouraging. Although terminal half-life of P61A6 is 5.6 hr, the high mean maximal concentration (Cmax) in plasma (7.1 µg/mL, or 12.2 µM) and the relatively high plasma concentration at 24 hr (6 µM), considering the in vitro IC50 value for P61A6 is approximately 2.2 µM (15, 18), might explain this significant inhibition, although further detailed pharmacological study is required.

One concern in developing GGTI is that inhibiting GGTase-I might lead to severe toxicity. Remarkably, we did not observe any severe toxic effects in mice. In addition, P61A6 has desirable in vivo pharmacokinetic properties, specifically clearance and distribution parameters that are suited to the small dosages required for efficacy. The pharmacokinetical profile of GGTI P61A6 obtained from this mice experiment is possibly different from that in human being; this should be addressed in the future clinical trial. One of the reasons for minimum toxicity observed with our GGTI compound is that we could achieve geranylgeranylation inhibition with low concentration of the compound compared with the study using other GGTI compounds, such as GGTI-2 (25). The difference may be due to the relatively long plasma half-life of our compound P61A6, which has a half-life of 5.6 h. GGTI-2418 was reported to have a half-life of 0.57 h (30). It is shown that inactivating the gene coding the β subunit of GGTase-I reduces lung tumor formation, eliminates myeloproliferative phenotypes, and increases survival rates of mice (12). Interestingly, in those studies, several cell types remained viable in the absence of GGTase-I, and myelopoiesis appeared to function normally (12). These observations are consistent with our results that the inhibition of GGTase-I can significantly inhibit tumor growth without inducing severe toxicity to mice.

In our previous in vitro study, we found that the treatment with GGTI P61A6 induced the inhibition of cell proliferation and cell cycle arrest at G1-phase accompanied by decreased S-phase, but not apoptosis, in various cancer cell lines, such as leukemic, breast cancer and pancreatic cancers (18). This cell proliferation inhibition was confirmed in the present in vivo study by the observation that treatment with P61A6 resulted in decreased BrdU-positive cells in the tumors, while apoptotic cells (TUNEL staining) were not affected by P61A6. We also checked the effect of P61A6 on tumor angiogenesis in the xenograft model, but did not find any obvious differences in the microvessel density in treated mice versus control. Taken together, these results suggest that the novel GGTI P61A6 inhibited tumor growth by inhibiting proliferation leading to the suppression of tumor growth. Theoretically, it is highly probable that combinations of apoptosis-inducing cytotoxic agents and this new identified GGTI compound will have a powerful anticancer effect on human cancers, and this possibility is being pursued now.

We have confirmed GGTase-I inhibition in tumors. We found that GGTI P61A6 impeded membrane association of geranylgeranylated proteins such as RhoA and Rap1. However, how this inhibition of membrane association of geranylgeranylated proteins by P61A6 contributed to the tumor growth inhibition remains to be determined, requiring future investigation. One possibility is that inhibition of RhoA function by blocking its membrane association, which negatively regulates expression of the Cdk inhibitor p21CIP1/WAF1, might partly be responsible for P61A6’s tumor suppression. Although other GGTI compounds, such as GGTI-298 and GGTI2166, were reported to inhibit PI3K/AKT2 pathway, leading to programmed cell death in human ovarian cancer cells (31), we observed neither apoptosis nor effect on phospho-Akt by our compound P61A6 (supplementary Figure S1). This difference of activation between our GGTI P61A6 and GGTI 298 and 2166 is interesting, and might due to the different effects on small G-proteins that our compound P61A6 inhibited their membrane association, while small G-proteins did not seem to be the targets of GGTI 298 or 2166 (31), although further investigation about the molecular mechanisms need to be done. In addition, treatment with GGTI P61A6 markedly inhibited the enzymatic activity of GGTase-I in tumor cells. Inhibition of GGTase-I activity in tumors was also observed with GGTI-2154 (15). Despite significant inhibition of membrane association of RhoA, we observed that tumors in treated groups kept growing, although much slower and smaller than that in the control group, even with the highest dosage. It was reported that dominant-negative forms of RhoA, Rac1, and Cdc42 only partially reverse Ras-induced malignant transformation (32, 33). This might partially explain the continuing growth of the treated tumors, although further investigations are certainly required to elucidate this phenomenon.

The intraperitoneal administration of GGTI P61A6 in the present study showed positive tumor-suppressing effects in SCID mice at doses that did not show adverse effects; thus we believe that intravenous administration should provide a better outcome for cancer therapy. Also, because this compound showed very good mucosa absorbance rates and long blood retention time, oral administration will be worth pursuing and good tumor-suppressing effects may be expected. Because GGTI P61A6 showed cell proliferation inhibition on other human cancer cell lines in addition to pancreatic cancer in our previous in vitro studies, it will be interesting to test various human cancer xenografts.

Taken together, the present study clearly demonstrates that our novel GGTI P61A6 inhibits GGTase-I activity, blocks protein geranylgeranylation, and induces tumor inhibition in human pancreatic cancer xenograft models at low doses, without inducing any obvious adverse effects. These findings prove that geranylgeranylation of proteins plays a critical role in Ras-driven tumorigenesis and provide strong evidence that GGTase-I is a feasible and promising molecular target for anti-cancer drug discovery.

Supplementary Material

Acknowledgements

We thank Luis Papa at the Division of Laboratory Animal Medicine of UCLA for his help on preparing and processing the tumor sections and Dr. Clara Magyar at the Tissue Procurement Core Laboratory of Department of Pathology and Laboratory Medicine at UCLA School of Medicine for significant help on tumor immunohistochemistry, imaging and analysis.

This work was supported by National Institutes of Health Grants CA32737 (to F. T.) and GM071779 (to O. K.) as well as by a grant from Susan E. Riley Foundation.

Abbreviations list

GGTase-I
protein geranylgeranyltransferase type I
GGTI
GGTase-I inhibitor
FBS
fetal bovine serum
FTase
Protein farnesyltransferase
FTI
FTase inhibitor
MVD
microvessel density
TUNEL
Terminal deoxynucleotidyl transferase dUTP nick end labeling

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