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* Istituto di Endocrinologia e Oncologia Sperimentale. I.E.O.S., CNR,
Laboratorio di Biochimica; I.R.C.C.S. "S. de Bellis" Castellana G. (Bari),
Dipartimento di Scienze Farmaceutiche, Università di Pisa,
Dipartimento di Biologia e Patologia. Cellulare e Molecolare "L.Califano," Università di Napoli "Federico II,"
|| Dipartimento di Scienze Farmaceutiche, Università di Salerno, Italy
1Correspondence: Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo 84084 Fisciano (Salerno), Italy. E-mail: maubiful{at}unina.it/chilaez@hotmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: cell growth • isoprenoid pathway
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. I6A is a component of mammalian serine tRNA, and it is conceivable that modified nucleoside plays an essential role for protein synthesis. In mammalian cells i6A is present unbound or bound to a 26 kDa protein; expression of the latter is correlated with cell proliferation (4)
. In plants, i6A and related compounds, called cytokinins, display hormone-like functions controlling cell division and differentiation in several tissues.
Previous studies by our group have demonstrated that i6A inhibits TSH-induced cAMP increase, DNA synthesis, I– uptake, and affects cAMP-dependent microfilament organization in thyroid cells (1)
, indicating that this molecule does interfere with this crucial cellular pathway. Moreover, it is known that i6A and its analogs show anti-neoplastic activity in vitro, where it can inhibit cell proliferation of several mammalian cell lines. i6A induces apoptosis in tumor cells, including myeloid and lymphoblastic cells lines (5
6
7
8
9
10
11
12
13)
.
The potential antitumoral activity of i6A, an adenosine and isoprenoid derivative, was observed in the early 1970s (5
6
7)
. Interest in the study of this compound has been reinforced by recent experimental observations that modified ribonucleosides induce apoptosis in human cell culture systems (11)
. It was then observed that the i6A anti-proliferative effect was due to CDK1 and CDK2 inhibition (14)
. However, the mechanism of action of the i6A is still unclear. The aim of the present study was to investigate the effects of i6A on the growth of both untransformed and transformed FRTL-5 cell line. The FRTL-5 transformed cell line, the KiMol cell line, was obtained by infecting the FRTL-5 cells with the Kirsten-Murine sarcoma virus (15)
. Moreover, the hypothesis that i6A could interfere with cell growth was tested in vivo in mice with xenograft tumors induced by KiMol cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells and culture
FRTL-5 thyroid cells (ATCC CRL 8305, rat thyroid cells) were grown in Coon’s modified Ham’s F12 medium supplemented with 5% calf serum. This cell line, derived from normal rat thyroid, retains in vitro the typical markers of thyroid differentiation, i.e., thyreotropin (TSH) dependency for the growth, capability to uptake iodine, synthesis and secretion of thyroglobulin (16
17
18
19)
. In addition, we have analyzed the i6A effects on the growth of a FRTL-5 transformed cell line, the KiMol cell line. This cell line was obtained by infecting the FRTL-5 cells with the Kirsten-Murine sarcoma virus (14)
. These cells express high levels of v-K-ras protein and no longer require TSH for growth. Moreover, KiMol cells have lost the thyroid differentiation markers and have acquired the capability to grow in semisolid medium and to induce the growth of tumors when injected in athymic mice (20)
. KiMol cells were grown in the same medium without hormones.
Cell proliferation assay
FRTL-5 cells were cultured in 100 x 15 mm tissue culture dishes (Falcon, Oxnard, CA, USA) and maintained in medium containing 5% serum, but no hormonal supplement (NoH) was added for 5 days. Then the medium was either maintained in NoH or supplemented with TSH 10–9 M, alone or in combination with different concentration of i6A at an established concentration for each experiment type (25–100 µM). After 24 h of exposure to i6A, the cycle cell was analyzed by flow cytometry. Cell growth in the presence of the various concentrations of each of the tested substances was measured in triplicate for each cell line.
In some experiments the cells were shifted for 72 h in a medium from which the hormonal supplement was removed (quiescent cells, NoH).
Incorporation of [3H]-mevalonate into cellular proteins.
Quiescent FRTL-5 cells were incubated with 10 µM lovastatin, 30 mCi/mL [5-3H]-MVA for 7 h. Density of cell culture ranged between 1.5 and 2.0 x 106 cells/mL in 100 mm Petri dishes. Cells were then washed three times with ice-cold phosphate-buffered saline (PBS), scraped from the dish and lysed in hypotonic buffer (10 mM Tris-HCl, pH 7.2; 1 mM phenylmethylsulfonylfluoride; 2 µM aprotinin; and 10 µM leupeptin), disrupted by sonication, and centrifuged at 3000 rpm for 10 min at 4°C. Equal amounts of each protein extract (
100 µg) were analyzed by 12% sodium dodecyl sulfate PAGE (SDS-PAGE) as described (21)
.
Western immunoblot analysis
Cell extracts from subconfluent cells grown in 100 mm Petri dishes were prepared. Cells were washed twice in PBS, scraped in PBS, and pelleted by centrifugation. Cell pellets were resuspended in lysis buffer (10 mM Tris-HCl, pH 7.2, 1 mM phenylmethylsulfonylfluoride, 2 µM aprotinin, and 10 µM leupeptin), disrupted by sonication, and centrifuged at 3000 rpm for 10 min at 4°C. Protein (50 µg) cell supernatants were electrophoresed on 12% SDS-PAGE, transferred to nitrocellulose membrane, and blocked with 7.5% milk in Tris-buffered saline-Tween 20 for 1 h at room temperature (22)
. The filters were then probed with lamin B polyclonal antibodies and farnesyl diphosphate synthase antibody (Santa Cruz Laboratories, Santa Cruz, CA, USA) at a 1:1000 dilution. Immunoreactive proteins were detected by incubation with horseradish peroxidase-conjugated donkey anti goat IgG (Bio-Rad, Hercules, CA, USA) by using the enhanced chemiluminescence system (ECL, Amersham, Buckinghamshire, UK).
Preparation of cytosolic and nuclear fractions
Cells were resuspended in 20 mM HEPES pH 7.5, 10 mM EDTA, 1 mM DTT, 300 mM sucrose, and protease inhibitors.
Intact cells and nuclei were removed by centrifugation at 1000 g for 10 min and supernatant was centrifuged at 10,000 g for 30 min (22)
. The nuclear fraction and the supernatant at 100,000 g containing the cytosol were analyzed by Western blot for the presence of lamin B.
Farnesyl diphosphate synthase assay
FPPs assay was carried out with some modification of the procedure of Krisans et al. (23)
and Gupta et al. (24)
. Briefly, cells were washed twice with 1 mL ice-cold PBS and scraped in 0.2 mL ice-cold lysis buffer (Imidazol 40 mM and DTT 50 mM). The cell lysate was centrifuged at 10000 rpm x 5 min and the supernatant was used for FPPS assay. FPPS was assayed in 150 µL containing 25 mM HEPES, pH = 7, 2 mM MgCl2, 1 mM dithiothreitol, 5 mM KF, 1% n-octyl-ß-glycopyranoside, 3.3 µM [4-14C] IPP (18 Ci/mmol), 3 µM unlabeled IPP, and 20 µM geranyl diphosphate. Reactions were started adding 40 µL of lysate containing 100 µg of total protein and incubated for 45 min at 37°C. Reactions were stopped by the addition of 150 µL 2.5 N HCl in 80% ethanol containing 100 µg/mL farnesol as a carrier. The samples were hydrolyzed for 30 min at 37°C to convert the FPP to farnesol and neutralized by the addition of 150 µL of 10% NaOH. The reaction product (farnesol) was extracted into 1 mL of n-hexane and an aliquot (200 µL) of the organic phase was used for radioactivity counting. One unit of enzyme activity is defined as the amount of enzyme required to synthesize 1 pmol of FPP/min. Parallel samples were assayed to evaluate the total and the nonspecific radioactivity. In all experiments, enzyme assays were carried out in duplicate. The coefficient percentages (CV%) of intra- and interassay variation were 3% and 4%, respectively.
Synthesis of fluorescent probe 1
The probe contained a dansylamino group (DNS) placed on the 2' portion of the sugar moiety of the adenosine structure. A C6 alkyl spacer between i6A and the DNS group, was inserted, since in analogous adenosine derivatives the spacer possessed optimal stereoelectronic features in order to avoid additional biological effects (25)
. The synthesis of compound 1 was accomplished as shown in Scheme 1
,starting from 2-O-(hexylphtalimido)-adenosine 2, which was prepared from commercially available adenosine as previously reported (26)
. N6-(3-methyl-2-butenyl)-2'-O-(hexylphtalimido)-adenosine 3 was obtained by alkykation of compound 2 with prenyl bromide (27)
. The phtalimido moiety was then removed with hydrazine [28]. The product, compound 4, was converted into final product 1 by treatment with dansyl chloride in the presence of triethylamine (28)
.
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N6-(3-methyl-2-butenyl)-2'-O-(phtalimidohexyl)-adenosine (3)
Prenyl bromide (0.80 mL, 6.9 mmol) was added to a solution of 2'-O-(hexylphtalimido)-adenosine 2 (0.904 g, 1.82 mmol) and BaCO3 (0.57 g, 2.9 mmol) in DMF (60 mL). The reaction mixture was stirred at room temperature in the dark for 48 h. DMF was then evaporated in vacuo and the residue subjected to chromatography on silica gel (CH2Cl2/MeOH 9:1) to afford 0.68 g (1.2 mmol) of pure compound 3 (66% yield).
N6-(3-methyl-2-butenyl)-2'-O-(aminohexyl)-adenosine (4)
A solution of N6-(3-methyl-2-butenyl)-2'-O-(hexylphtalimido)-adenosine 3 (0.79 g, 1.4 mmol), 95% ethanol (30 mL) and hydrazine (0.75 mL, 2.4 mmol) was stirred for 16 h at room temperature. The reaction mixture was then filtered and the filtrate concentrated in vacuo. The residue was subjected to chromatography on silica gel (CH2Cl2/MeOH 9:1) affording 0.37 g (0.85 mmol) of pure compound 4 (60% yield).
N6-(3-methyl-2-butenyl)-2'-O-(dansylaminohexyl)-adenosine (1)
Dansyl chloride (0.25 g, 0.93 mmol) was added to a solution of N6-(3-methyl-2-butenyl)-2'-O-(aminohexyl)-adenosine 4 (0.37 g, 0.85 mmol) and triethylamine (0.22 mL) in dry chloroform (30.0 mL) and the reaction mixture was stirred overnight in the dark at room temperature. Chloroform was evaporated in vacuo and the residue purified by chromatography on silica gel (CH2Cl2/MeOH 9:1) affording 0.042 g (0.063 mmol) of pure compound 1 (7.4% yield). 1H-NMR (CDCl3, 200MHz) 
1.08–1.62 (m, 8H), 1.72 (br s, 6H), 2.68–2.91 (m, 2H), 2.88 (s, 6H), 3.16–3.50 (m, 2H), 3.65–3.84 (m, 1H), 3.90–4.06 (m, 1H), 4.10–4.32 (m, 2H), 4.32–4.39 (m, 1H), 4.46–4.52 (m, 1H), 4.75 (dd, 1H, J=7.6, 4.6 Hz) 5.30–5.45 (m, 1H), 5.79 (d, 1H, J=7.7 Hz), 5.96 (br, 1H), 6.51 (br, 1H), 7.17 (d, 1H, J=7.5 Hz), 7.46–7.56 (m, 2H), 7.74 (s, 1H), 8.22–8.49 (m, 3H), 8.53 (d, 1H, J=8.4 Hz); MS m/e 668 [M+H]+, 335 [M-dansylaminohexyl +H]+ or [dansylaminohexyl +2H]+.
Immunofluorescence
FRTL-5 cells grown on coverslip glasses were incubated for 24 h in culture medium containing 5% calf serum in the presence or in the absence of 100 µM i6A labeled with a fluorescent probe. The cells were then fixed with 3.7% paraformaldehyde. Coverslips were mounted on 50% glycerol in PBS and examined by fluorescence microscopy.
Tumorigenicity assay
All experiments were performed in 6-wk-old male athymic mice. Animals were injected (day 0) on the dorsal right side with a suspension of 0.2 mL containing 1· 10 6KiMol cells, and the site of injection was marked. At day 3 animals were divided in two groups, control and i6A were dissolved in 0.2 mL of sterile saline solution (0.9% NaCl) and injected subcutaneously (s.c.) at the previous injection site. Before injection, the solutions were sonicated to facilitate the solution of lipophilic components. Saline solution was used for the injection of the control group. Treatment was repeated at 72 h intervals. Tumor diameters were measured with callipers every other day until the animals were killed. Tumor volumes (V) were calculated by the formula of rotational ellipsoid: V = A · B 2 /2 (A=axial diameter, B=rotational diameter). After 20 days in the control group, tumor burden was exceeding 10% of the host weight; therefore, according to the regulation for animal welfare the experiment was stopped, the animals were killed, and the tumor weight was evaluated. During the treatment, none of mice showed signs of wasting or other visible indications of toxicity. Furthermore, the dose of i6A showed no detectable reduction of the spontaneous activity, as we observed unimpaired locomotion of the treated mice. All mice were maintained at the Biology and Pathology Animal Facility Department, and all animal studies were conducted in accordance with the Italian regulation for the welfare of animals in experimental neoplasia (29)
.
Other assays
Proteins concentration was determined by a colorimetric method and the reagent was obtained from Bio-Rad; recrystallized bovine serum albumin was used as standard.
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RESULTS |
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, upper panel). Enhanced inhibitory effect on the cell growth was observed in transformed KiMol cells treated with i6A at the same concentrations (Fig. 1
, upper panel and Table 1
).
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I6A inhibiting effect on cell growth was reverted by the co-addition of 0.1 µM farnesyl pyrophosphate (FPP), but not of mevalonic acid (MVA) (Table 1)
. The addition of cholesterol, dolichol (data not shown), FPP, or MVA (Tab 1)
did not elicit any effect.
Down-regulation of expression and activity of FPPS in FRTL-5 and KiMol cells
Since i6A effect was reverted by FPP, a key molecule of prenylation pathway, the expression and activity of farnesyl transferase (FTase), farnesyl pyrophosphate synthase (FPPS), and mevalonate kinase (MVKase) in FRTL5 and KiMol cells treated with i6A 100 µM for 24 h were investigated. The FPPS expression was found to be strongly decreased in i6A-treated KiMol cells and completely inhibited in i6A-treated FRTL-5 cells (Fig. 1A
). The FPPS activity significantly decreased in KiMol cells (Fig. 1B
).
Expression of two other enzymes of the isoprenoid pathway tested, such as mevalonate kinase and farnesyltransferase, was unaltered (data not shown). The inhibition of cell growth exerted by i6A appears to be due to inhibition of FPPS, reinforcing the role of isoprenoids in the regulation of cellular metabolic processes.
Effects of i6A treatment on protein prenylation
Because the farnesyl pirophosphate is required for the protein prenylation, we analyzed the prenylated proteins in i6A-treated cells. In FRTL-5 cells, metabolically labeled with [3H]mevalonate for 24 h, radiolabeled prenylated proteins in cell lysates are separated by electrophoresis into major bands of 21 to 26 kDa (representing mostly geranylgeranylated small GTP binding proteins, but also farnesylated Ras proteins) and 45 to 90 kDa bands in agreement with earlier observations by our group (21
, 22)
. FRTL-5 cells treated with i6A at 100 µM showed a reduction into the incorporation of the [3H]-mevalonate into cellular proteins. Cells exhibited a substantial decrease in the intensity of 68, 46, 34 kDa, whereas a minor reduction in the intensity of the 26 to 21 kDa protein bands occurred (Fig. 2
A). The change in protein prenylation was not induced by a decrease in protein synthesis (data not shown). Among the most affected prenylated proteins by i6A, attention was focused on the 68 kDa protein band, which was identified as lamin B, a protein involved in integrity of the nuclear membrane. To verify whether the inhibition of protein prenylation by i6A could induce the translocation of this protein from nuclear membrane to cytosol fraction, the localization of lamin B was investigated by Western blot. As shown in Fig. 2C
in untreated cells, intact lamin B was present only in the nuclear fraction. Conversely, in i6A-treated cells lamin B was also found in the cytosolic fraction, suggesting that inhibition of prenylation affected the localization of lamin B, thus inhibiting its biological activity.
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Analysis of the mechanism underlying i6A effect
To verify whether i6A effect on cell proliferation was mediated by adenosine receptor, the effects of i6A and unmodified adenosine on the growth of FRTL-5 and KiMol cells were studied. As shown in Table 1
, adenosine had no effect on cell proliferation inasmuch as 10 nM toxin pertussis able to interrupt a signal transduction system by the ADP-ribosylation of G-proteins did not reverse the i6A action (Table 1)
(30
, 31)
. Strictly correlated to these effects on cell proliferation, we observed that adenosine did not affect FPPS expression or protein prenylation (Fig. 1A
, 2B).
To investigate whether i6A comes inside the cells, we designed and synthesized a fluorescent probe containing a dansylamino moiety attached to the i6A structure. Before proceeding with the experiments, its biological activity was proved to be similar to that of i6A (data not shown). FRTL-5 cell were then incubated with 100 µM modified i6A for 1 h, fixed, and analyzed by fluorescence microscopy. As shown in Fig. 3
A, the labeled i6A was internalized in the cytoplasm.
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To verify a direct effect of i6A on FPPS enzyme, an in vitro assay of FPPS activity on cellular protein extracts was performed. i6A inhibited FPPS activity in FRTl-5 and more efficaciously in KiMol cells in a dose-dependent manner (Fig. 1C
).
N6-Isopentenyladenosine reduced the tumor volume induced in vivo
Since i6A significantly inhibited the growth of the transformed KiMol cell lines, we studied the effects of i6A on the growth of xenograft tumors induced by KiMol cells. Cells were injected s.c. into the flank of 30 athymic mice. After 15 days when tumors were clearly detectable, i6A was injected in the peri-tumoral area on days 2 and 5 of a 7-day cycle for three cycles. i6A treatment (0.5 mg/kg/dose) induced a significant reduction in tumor weight (80%) with respect to the control mice. No detectable toxic or hypolocomotor effects on the treated animals were observed (Fig. 3B
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 22
, 32
33
34)
, suggest new therapeutic strategies interfering with p21ras activation and affecting the isoprenoid pathway. In addition to chemically synthesized compounds, much effort has been directed to identifying endogenous mechanisms of tumor suppression and, by high-throughput screening, of anticancer natural products. Some of these compounds have been discovered. However, several issues in the research remain unsolved. For example, the mechanism by these inhibitors suppress tumor growth is still unclear. In the present work, we show that the exposure of FRTL-5 and KiMol thyroid cells to i6A leads to a cell cycle arrest. The inhibition of cell growth exerted by i6A appears to be due to a strong inhibition of the FPPS activity, without any involvement of other enzymes of the isoprenoid pathway. The antiproliferative effects of i6A were reversed by the addition of FPP; treatment with mevalonate or other isoprenoid products did not affect cell growth. i6A down-regulates FPPS expression directly, without any mediation by the adenosine receptor. In fact we showed that i6A is internalized in FRTL-5 cells, and as a result modulates FPPS activity, as demonstrated by an in vitro assay of its activity on cellular protein extracts. Then, i6A seems to exert its effects through a feedback mechanism on the FPPS; whether these effects are mainly related to the increased degradation rate rather than to a modified activation state of the enzyme, remains to be elucidated.
I6A inhibits FPPS activity more efficaciously in KiMol cells than in normal FRTL-5, and this finding can explain why transformed cells are more sensitive to this new prenylation inhibitor. The FPPS inhibition causes in the cell a reduction of FPP and of its downstream product geranylgeranylpyrophosphate (GGPP), both essential for the activity of the prenylated proteins. We reported that i6A inhibited the incorporation of (3H)-mevalonate into cellular prenylated proteins of thyroid FRTL-5 cells (21)
. We previously described that the thyroid cells incorporated (3H)-mevalonate into cellular proteins corresponding to the molecular masses of 21–26, 34, 46, 68 kDa (22)
. The cluster of proteins of 21 to 26 kDa are likely to be mainly GTP binding proteins, whereas the 68 kDa correspond to nuclear lamin B, a protein implicated in the cell proliferation (38). These proteins play an important role in a wide variety of cellular processes such as the transduction of growth-modulating signals, intracellular protein traffic and secretion (35
, 36)
. We observed that i6A specifically removes lamin B from the nuclear membrane compartment, suggesting that the arrest in G0-G1 is probably induced by the decrease of mevalonate-derived isoprenoid groups bound to this nuclear membrane protein. In addition, we observed a decreased labeling of the 21 to 26 kDa proteins in i6A-treated FRTL-5 cells with respect to proliferating KiMol cells.
Finally, we have evaluated the i6A effect in tumor xenografts showing a significant growth inhibition and no toxic effect, supporting its antineoplastic activity once more.
In the present study we have confirmed the inhibitory role of N6-isopentenyladenosine on cell proliferation. Moreover, we have highlighted that N6-isopentenyladenosine inhibits FPPS and protein prenylation and affects lamin B processing. Our findings indicate that this isoprenoid end product might be used for antineoplastic therapy, an application emulating that of the lovastatin and/or prenylation inhibitors (37)
. The objective of this study was to set the development of this new prenylation inhibitor and to focus on the issues surrounding development of signal transduction inhibitors. The emphasis on this small isoprenoid end product is that it could represent, compared with the other prenylation inhibitors, a novel endogenous suppressor of cancer growth.
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ACKNOWLEDGMENTS |
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Received for publication June 8, 2005. Accepted for publication September 22, 2005.
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) and KiMol (
) cells in the G0-G1 phase and reduction in the S phase of the cell cycle by i6A at 25, 50, and 100 µM. Samples were collected at 24 h in presence of different concentrations of i6A, and analyzed for DNA content by flow cytometry. Results are presented as mean ± SD from 4 independent experiments (*P<0,05 vs. control group). Lower panel: i6A inhibits FPPS expression and activity in thyroid cells. A) i6A 100 µM reduces the FPPS expression in KiMol cells and it is completely inhibited in FRTL-5 cells with respect to control. B) i6A 100 µM significantly inhibits FPPS activity in KiMol cells (*P<0.05, paired t test). C) the FRTL-5 and KiMol homogenates (100 µg) were treated with i6A concentration from 10 µM up to 100 µM for 30 min at 37°C, then FPPS activity assay was performed. Bars represent the mean ± SD from 3 separate experiments. (*P<0.05)
