| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,1
* Dipartimento di Biologia Molecolare;
Dipartimento di Medicina Clinica e Scienze Immunologiche, Policlinico Le Scotte;
Dipartimento Farmaco Chimico Tecnologico;
Dipartimento di Chirurgia, Sezione di Biologia Applicata, Policlinico Le Scotte, Università degli Studi di Siena, Siena, Italy;
|| Dipartimento di Scienze Farmaceutiche, Università degli Studi di Genova, Genova, Italy; and
¶ Dipartimento di Biologia di Base ed Applicata, Università degli Studi dell’Aquila, Coppito L’Aquila, Italy
1Correspondence: A.S., Università degli Studi di Siena, Dipartimento di Biologia Molecolare, via Fiorentina 1, 53100 Siena, Italy. E-mail: santucci{at}unisi.it; M.B., Università degli Studi di Siena, Dipartimento Farmaco Chimico Tecnologico, via A. Moro, 53100 Siena, Italy. E-mail: botta{at}unisi.it
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Key Words: bone cells • apoptosis • bone cancer • osteoblast • tyrosine kinase
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
, 2)
. Clinical research of new pharmacological approaches for osteosarcoma treatment has moved in several directions: new drug research, more intensive treatments, immunotherapy, and gene therapy. Although the prognosis of osteosarcoma has been significantly improved by the adoption of multiagent chemotherapy, a considerable number of patients die (3
4
5
6
7
8)
. The Src family of tyrosine kinases is an important mediator of cell growth factors and mitogenesis and thus is currently targeted by experimental antimitotic drugs. Selective inhibitors of c-Src function represent an important field of research for treatment of tumors, since Src phosphorylation inhibition may stop uncontrolled tumor growth (9)
. During the past decade, many tyrosine inhibitors have been described, some of them belonging to the pyrazolo[3,4-d]pyrimidines class. A series of new pyrazolo[3,4-d]pyrimidine derivatives has been recently synthesized that proved to have antiproliferative activity in A431 cells (10
11
12)
, ductal infiltrating carcinoma (13)
, prostate carcinoma (14)
, squamous carcinoma (15)
, and leukemia cells (16)
.
Although Src kinase is involved in many fundamental cellular processes, including proliferation, migration, and survival, to date there is no report on Src overexpression or Src-related gene mutation in osteosarcoma cells. Nevertheless, a high Src expression has been found in mouse osteoclasts, where it is implicated in the regulation of cell growth, migration, and survival (17)
. That is why Src inhibitors have also been studied for their potential activity in bone resorption for the therapy of osteoporosis in rodent models (18
19
20)
. Moreover, the involvement of Src has been proven to be crucial in the process of differentiation of mouse osteoblasts (21)
. An elevated activity of Src family kinases was demonstrated in high-metastatic human osteosarcoma sublines (22)
. Recently, authors (23)
reported that the regulation of c-Src kinase activity seems to be involved in the development of the osteosarcoma malignant phenotype.
In the present study, we assayed in human osteosarcoma SaOS-2 cells the potential activity of novel pyrazolo[3,4-d]pyrimidine derivatives with respect to the well-known lead compound PP2. First, we screened the novel compounds for their inhibitor activity in cell viability. By means of terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL), Hoechst, and flow cytometric assays, two compounds were shown to possess higher antiproliferative and proapoptotic activities as compared with PP2. Specifically, a new compound, namely SI-83, not only inhibited c-Src phosphorylation and proved to be active in five human osteosarcoma cell lines but also significantly decreased in vivo osteosarcoma tumor mass in a mouse model and additionally showed low cytotoxicity in primary human osteoblasts.
Taken together, these results suggest that new pyrazolo[3,4-d]pyrimidine derivatives may provide therapeutic benefit by preventing the growth of bone sarcomas with potentially low side effects in the nonneoplastic counterpart cells.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Bone samples were obtained from five male patients (average age: 55 yr old) who underwent total hip replacement surgery. The patients were selected to exclude those who have received a previous therapy with hormone replacement or glucocorticoid treatment during the previous 2 yr. Trabecular bone fragments were extensively washed in phosphate buffered saline (PBS; Sigma-Aldrich) to remove blood and bone marrow and then explanted into culture containing DMEM supplemented with 10% (vol/vol) FCS, 2 mM L-glutamine (Life Technologies, Invitrogen, Carlsbad, CA, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin. Cultures were carried out as reported previously (24
, 25)
. The cells obtained with this method were positive for alkaline phosphatase activity and expression of osteoblast differentiation markers (24
, 25)
.
Cell treatments
Pyrazolo[3,4-d]pyrimidine derivatives (26)
were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at various concentrations and diluted in cell culture medium before use. In parallel, PP2 (Calbiochem, Darmstadt, Germany) was used as a reference compound. Controls were carried out with DMSO concentrations corresponding to the higher doses of the test compounds. The final DMSO concentration did not exceed 0.2% (vol/vol) and did not affect the parameters analyzed. Cells were treated when at confluence.
Cell viability assays
Cell viability was quantified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Molecular Probes, Invitrogen). Cells (
3x104 for each well) were seeded in 96-multiwell plates with culture medium and then exposed to compounds at concentration of 25 µM for 48 h. The medium was removed, and the cells were incubated for 4 h with fresh medium in the presence of 1.2 mM MTT. After solubilization in DMSO, the absorbance of the formazan dye was measured with a microplate absorbance reader at 540 nm. The inhibitory activity of the compounds was compared with that of PP2 used at the same concentrations of compounds.
To evaluate half-maximal inhibitory concentration (IC50) for S-7, SI-83, and PP2, cells were assayed by MTT and the percentage of inhibition of cell viability at various concentrations (1–100 µM) for 48 h was evaluated.
Cell viability was quantified by MTT in human osteoblasts. Approximately 30,000 cells were seeded in a 24-multiwell plate, grown for 7 days (confluence), and then exposed for 48 h to compounds at concentrations equal to their respective IC50, calculated in SaOS-2 cells as reported above.
DNA duplication was assessed using a colorimetric immunoassay kit to quantify the incorporation of 5-bromo-2'-deoxyuridine (BrdU) during DNA synthesis (Roche Applied Science, Mannheim, Germany). This assay was performed on cells previously treated for 24 h with compounds S-7 and SI-83 at 12.5 and 25 µM concentrations, respectively. Briefly, BrdU was added to the cells for 18 h before the end of the treatment. Fixation and partial DNA denaturation were performed before staining with anti-BrdU antibody. Immune complexes were detected by subsequent substrate reaction, and the absorbance was measured using a multiwell spectrophotometer (Bio-Rad, Hercules, CA, USA).
Apoptosis and cell cycle assays
The DNA strand breaks in the apoptotic cells were assayed by the TUNEL in situ cell death detection kit (Roche Applied Science). SaOS-2 cells were grown on coverslips placed in sterile shell vials (Sarstedt, Nümbrecht, Germany) and treated with PP2, SI-83, and S-7, as well as with DMSO as a negative control, at a concentration of 12.5 µM for 48 h. The TUNEL assay was then performed according to the manufacturer’s instruction. Pictures were taken, and apoptotic nuclei were counted. For each sample, five independent pictures were taken.
The percentage of apoptotic cells was evaluated by FACS analysis. Then, 1 x 106 SaOS-2 cells were plated in 6-well multiplates and grown in complete medium until confluence. Two different treatments were done for SaOS-2 and for primary osteoblasts. SaOS-2 were treated for 48 h with compounds at concentrations of 3, 25, and 100 µM, and IC50. Primary osteoblasts were treated at concentration equal to IC50 calculated in SaOS-2 cells as described for 24, 48, and 96 h. After treatment, cells were washed three times in PBS and fixed overnight in ice-cold 70% (vol/vol) ethanol at –20°C. The cell suspension was centrifuged, washed twice with 1 ml of PBS, and resuspended in 1 ml of PBS containing ribonuclease (Type-1A, 1 mg/ml; Sigma-Aldrich) and propidium iodide (PI, 50 µg/ml; Sigma-Aldrich). The tubes were placed on ice in the dark until the cellular orange fluorescence of PI was collected in a linear scale using a FACS-calibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) equipped with an excitation laser line at 488 nm and a 575 ± 15 nm band-pass filter. At least 20,000 events were collected for each sample using the Cell Quest software (Becton-Dickinson) and the pulse processing module for doublet discrimination; debris were excluded from the analysis by an appropriate morphological gate of forward scatter vs. side scatter.
Hoechst staining was used to evaluate healthy, necrotic, and apoptotic cell morphology. Approximately 2 x 105 cells were seeded on eight chamber polystyrene vessels (BD Falcon, Bedford, MA, USA), grown in complete medium until confluence, and then exposed to PP2, SI-83, and S-7 at concentration of 25 µM for 48 h. Then cells were fixed for 15 min in 4% paraformaldehyde, washed in PBS, air dried, and incubated for 10 min with 10 µg/ml Hoechst 33342 (Invitrogen, Paisley, UK), a bisbenzimide cell-permeant dye that fluoresces bright blue on binding to DNA. The coverslips were rinsed 5x in distilled water and allowed to air dry in the dark. Coverslips were then mounted using PBS/glycerol and examined by fluorescence microscopy and digital image capture.
Immunoblotting
For Phospho-SrcY416 detection, cells were lysed in ice-cold 0.1% sodium dodecyl sulfate (SDS) containing a cocktail of protease inhibitors and 1 mM sodium orthovanadate (Sigma-Aldrich); 10–20 µg of cell cultures protein lysates, diluted in reducing buffer, was resolved by 12% SDS-PAGE and electrotransferred onto nitrocellulose. All antibodies used were from Cell Signaling Technology (Denver, CO, USA). Proteins were probed with primary antibodies o.n. at 4°C, followed by secondary antibodies for 1 h at room temperature. Detection was obtained by ImmunoStar HRP (Bio-Rad). Band areas were detected using ImageScanne (Amersham Bioscience, Little Chalfont, UK), and the optical densities of the band were analyzed by Image Master software (Amersham Bioscience). Band optical densities of Phospho-SrcY416 were normalized against those of total Src as an internal control.
Nodules formation and mineralization
The mineralized nodule formation and their degree of mineralization were determined for osteoblasts grown in 6-well plates for 7, 14, and 21 days using alizarin red S staining. Cells were treated with PP2 and SI-83 at their respective IC50, IC20, and IC5, calculated in SaOS-2 cells. The compound administration was repeated every 3 days in correspondence with medium changes. Briefly, after two washes with PBS, cells were fixed with ice-cold 70% (vol/vol) ethanol for 1 h, washed again, and stained with 40 mM alizarin red S (Sigma-Aldrich) in deionized water (adjusted to pH 4.2) for 10 min. Cells were rinsed with PBS and destained for 15 min with 10% (wt/vol) cetylpyridinium chloride in 10 mM sodium phosphate (pH 7.0). The extracted stain was transferred to a 96-well plate, and the absorbance at 562 nm was measured using a plate/reader spectrophotometer.
Biochemical alkaline phosphatase activity assay
The ALKP activity was measured directly on monolayer cultures. The medium was removed, and the cells were washed three times with PBS and shaken for 30 min at 37°C in 1 ml of 10 Mm p-nitrophenilphosphate (Sigma). The p-nitrophenylphosphate solution was removed, and the reaction was stopped by adding 1 ml 1 N NaOH. The optical density was measured at 405 nm. The ALKP activity values were normalized to the relative number of viable cells as determined in 12-well plates using the above-mentioned proliferation assay.
SaOS-2 xenograft
CD1 nude mice (Charles River, Milan, Italy) were maintained under the guidelines established by our institution (University of L’Aquila, Medical School and Science and Technology School Board Regulations, complying with the Italian government regulation n.116 January 27, 1992 for the use of laboratory animals). Before tumor cell implantation, mice were anesthetized with a mixture of ketamine (25 mg/ml)/xylazine (5 mg/ml). Xenografts were obtained by subcutaneously injecting 1 x 106 SaOS-2 cells in 100 µl of 12 mg/ml Matrigel (Becton-Dickinson). Mice received daily the drug by per os administration at the doses indicated. Tumor growth was monitored daily, and at the endpoint after 26 days, the tumors were excided, weighed, and processed for histology. Briefly tumors were fixed in 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2, and embedded in paraffin. Slide-mounted tissue sections (4 µm thick) were deparaffinized in xylene and hydrated serially in 100, 95, and 80% ethanol. Endogenous peroxidases were quenched in 3% H2O2 in PBS for 1 h, and then slides were incubated with anti-human Src (pY416) phosphospecific antibody (Biosource, Camarillo, CA, USA) for 1 h at room temperature. Sections were washed three times in PBS, and antibody binding was revealed using the Sigma fast 3,3'-diaminobenzidine tablet set (Sigma).
Statistics
Data are means ± SE of three independent experiments performed in duplicate. Statistical analysis was performed by the unpaired Student’s t test. A value of P < 0.05 was conventionally considered as statistically significant.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
. Further details are reported in Supplemental Data.
Novel pyrazolo[3,4-d]pyrimidines affect cell viability of SaOS-2
A set of new pyrazolo[3,4-d]pyrimidine derivatives (Table 1
) was assayed to evaluate their potential inhibition of cell viability toward human osteosarcoma SaOS-2 cells. The compounds were tested by MTT assay at a concentration of 25 µM, and their effects were evaluated after 48 h (Fig. 1
A) on the basis of previously unreported results. PP2 was tested as well in SaOS-2 and caused an inhibition of cell viability of 63.5% with respect to vehicle (DMSO). The most active compound was SI-83, inhibiting cell viability by 85% and showing a significantly higher activity than PP2. S-7 and SI-10 induced an inhibition of 69 and 66%, respectively.
|
|
On the basis of such results, we chose SI-83 and S-7 as the elective molecules to perform the subsequent experiments. By BrdU incorporation assay, we proved their antiproliferative activities toward SaOS-2. In fact, after the treatment with PP2, SI-83, and S-7 at two different concentrations, we observed that new DNA synthesis was blocked, with SI-83 being the most active molecule (Fig. 1B
). Then, by MTT assay, both SI-83 and S-7, as well as PP2, showed a dose-dependent inhibitory capacity of cell viability (Fig. 2
, top panels), although apparently with a different potency. The kinetics of induced inhibition seemed to be different for the compounds (Fig. 2
, bottom panels). In fact, while in the cases of PP2 and S-7 the inhibitory activity gradually increased from zero up to the highest values following a linear trend, SI-83 was apparently less active at very low concentrations, but once it reached a threshold concentration (5 µM), its activity increased very rapidly. In particular, at a concentration of 25 µM, SI-83 was able to inhibit cell viability of 83%, a percentage of inhibition obtained by PP2 at concentrations >50 µM and never reached by S-7. IC50 values calculated for the three compounds resulted in 8.07 µM for PP2, 14.55 µM for S-7, and 12.64 µM for SI-83. Although on the basis of IC50 PP2 seemed the more effective molecule, nevertheless, this ranking criterion may be reductive since it does not take into account the maximum value of inhibition obtained for each single compound nor the different kinetics. In fact, SI-83 at its highest potency of action inhibited cell viability by 93.3%, much more than PP2 (85.3%) and S-7 (71.1%). The different curve profiles of the activity of the three compounds may be due to different affinities of these molecules for their target Src. SI-83 had a less steep initial trend, showing for dosages >5 µM rapidly increasing activity and reaching its maximum of activity already at a concentration of 50 µM. These data seem to indicate that SI-83 is a better compound than PP2 and S-7 in inhibiting cell viability and that the IC50 value may not be sufficient to express a qualitative ranking of bioactive compounds if dose dependence and the maximal value of their activity are not taken into account.
|
SI-83 pyrazolo[3,4-d]pyrimidine inhibits Src-Y416 phosphorylation in SaOS-2
To verify if SI-83 decreased the Src phosphorylation in drug-treated cells, we performed Western blotting with anti-non-phospho- and anti-phospho-SrcY416 antibodies. The treatment with both PP2 and SI-83 strongly inhibited the phosphorylation of Src at Y416 by 50 and 55%, respectively (Fig. 3
A, B).
|
Molecular modeling simulations allowed us to shed light on the possible binding mode of SI-83 into the Src kinase structure. The best docked conformation of this molecule into the ATP-binding pocket showed hydrophobic contacts between the m-chlorophenylamino side chain at C4 and the hydrophobic region II of Src, as well as interactions of its methylthio substituent at C6 and Ala390 (Fig. 3C
). Finally, part of the N1 side chain was located within the adenine pocket, while its terminal portion pointed toward the hydrophobic region I. Such theoretical results suggested that hydrophobic contacts with both the hydrophobic region I and II could play a pivotal role in determining the affinity for Src.
These results suggest that cell proliferation and induction of apoptosis are modulated by Src activity in SaOS-2 and that antiproliferative and proapoptotic activities of SI-83 work with a molecular mechanism analogous to that of PP2, although potentiated in SI-83, probably due to its molecular structure.
Src is not overexpressed in osteosarcoma, and Src overexpression by itself is not sufficient for oncogenic transformation (37)
. More generally, even when present, like in colon cancers, Src mutations do not seem to be the predominant mechanism of Src activation in tumors (38
39
40)
. On the other hand, some findings suggest that enhanced activity of Src family kinases and hyperphosphorylation of paxillin synergistically contribute to the high metastatic potential of osteosarcoma (22)
, although a paxillin-independent scenario has also been suggested (37)
. An Src role in osteosarcoma has been linked in U2-OS and SaOS-2 cell lines to the antioncogenic role of CD99 through the regulation of caveolin-1 and the inhibition of Src activity (23)
. Very recently, it has been reported that Src activation/phosphorylation levels do not correlate with the osteosarcoma cell line response to dasatinib, suggesting that low levels of Src kinase activation are sufficient to induce biological properties (41)
.
Pyrazolo[3,4-d]pyrimidines induce apoptosis in SaOS-2
Specific proapoptotic actions have been reported for Src inhibitors (13)
. To evaluate if PP2 and the new derivatives could exert a proapoptotic effect in SaOS-2, a TUNEL assay was performed after a 48 h treatment with different concentrations of PP2, SI-83, and S7. For all three compounds assayed, a proapoptotic effect was evident. The data indicated that, as observed for other cell types (10
, 11)
, PP2 also induced apoptosis in SaOS-2 cells (Fig. 4
), probably as a consequence of Src signaling inhibition. Analogously, S7 induced apoptotic cell death, since its effect was more evident than that of PP2 (Fig. 4B
). The strongest proapoptotic effect seemed that of SI-83, also in consideration of the different IC50 of the compounds (Fig. 4A, B
).
|
We performed flow cytometric assays on SaOS-2 cells, after a treatment with PP2, S7, and SI-83 at different concentrations. As reported, Src is involved in cell cycle progression and in proceeding from G0/G1 to subsequent phases (27)
. The compounds tested induced apoptosis, as represented by the presence of G0 cell subpopulation (Fig. 4C
). PP2 was able to stop cell cycle progression in G0 and G1 phases in a dose-dependent manner, as observed in other cell types (10
11
12
13
, 28
29
30
31
32
33
34
35)
. Moreover, the G0/G1 phase was normal, while the sub-G0/G1 phase rose as the compound concentration incremented, and we observed a complete abolition of phases S and G2/M at its maximum value. The situation was different for cells treated with S7 at every concentration, where the treatment induced an arrest of cell cycle progression in the G2/M phase. SI-83 showed a PP2-like behavior, although in this case the abolition of phases S and G2/M was already appreciable at its IC50. SI-83 induced an arrest of cell cycle progression in a dose-dependent manner and proved to be the most proapoptotic compound.
Since cytotoxicity may occur as an undesirable side effect of compounds with a potential therapeutic application and may contribute to a diminished cell proliferation, we evaluated cell morphology changes by the Hoechst assay. As shown in Fig. 4D
, PP2- and SI-83-treated cells revealed morphology similar to that of control even if condensed apoptotic nuclei were visible as well. On the contrary, S7-treated cells showed nuclei malformation, probably due to DNA damage. Taken together, these results suggest that Src inhibitors are able to arrest cell growth and induce apoptosis. However, while S7 exerts its action through DNA damage, SI-83 and PP2 act by increasing apotosis.
Although it is currently impossible to directly correlate the inhibition of Src phosphorylation to the induction of apoptosis, since many effectors downstream to Src are still to be elucidated, especially in human osteoblasts and osteosarcoma, our results support the hypothesis of a role of Src in affecting cell viability, proliferation, cell cycle progression, and apoptosis in human osteosarcoma cells. This is in line with what has been reported by others (36)
who showed an indirect link of Src and phosphoinositide 3-kinase/Akt in the control on growth arrest and apoptosis in SaOS-2 cells. Our findings are also in agreement with what was recently reported by Diaz-Montero et al. (37)
on the role of Src in anoikis resistance of human osteosarcoma cells.
It is well known that Src is the first member of a kinases family broadly controlling cell cycle, proliferation, differentiation, and survival. In this respect, no Src inhibitor is expected to have a total specificity. In our case, the involvement of additional phosphorylations beyond Src is suggested also by the inhibition of the total protein phosphorylation levels induced by pyrazolo[3,4-d]pyrimidine derivatives (data not shown).
In primary human osteoblasts SI-83 pyrazolo[3,4-d]pyrimidine less affects proliferation and apoptosis
Expression of Src and related kinases is important in many physiological processes; thus, inhibition of Src could also result in considerable adverse effects in normal cells. For this reason, we also tested SI-83, the most active compound in SaOS-2 cells, for its inhibitory activity in cell viability of primary human osteoblastic cells by MTT assay. For osteoblast treatment, both SI-83 and PP2 were used for 48 h at the same concentrations adopted for SaOS-2 cells. The inhibitory activity of PP2 in osteoblasts was quite similar to that observed in SaOS-2 cells (Figs. 5
Aand 1A
). In contrast, SI-83 showed an opposite effect. In fact, when this compound was used at 25 µM, the percentage of cell viability was 80% (65% higher with respect to that observed in SaOS-2), while when it was used at 12.5 µM (close to its IC50), osteoblast viability was not affected at all. These data suggest that SI-83 may be selective for transformed cells.
|
To better evaluate the proapoptotic activity in osteoblasts, we also performed a time-course cytofluorimetric analysis, using PP2 and SI-83 at their respective IC50, as previously determined in SaOS-2 cells (Fig. 5B
). Although an induction of apoptosis was observed, nevertheless, SI-83 induced half of apoptosis in osteoblasts with respect to PP2. More importantly, when the respective same concentrations (IC50) and times of treatment (48 h) were compared between SaOS-2 and osteoblasts (Figs. 3C
and 5B)
, we could observe a decreased proapoptotic effect in osteoblasts with respect to SaOS-2. Finally, it is noteworthy that induced apoptosis was very similar at 48 and 96 h. Hoechst staining confirmed that treatment with SI-83, as opposed to treatment from PP2, did not alter cell morphology or cause DNA damage (Fig. 5C
). These remarkable results strongly suggest a different action of SI-83 in primary osteoblasts with respect to osteosarcoma cells.
Our findings in primary human osteoblasts are in agreement with previous reports that osteoblasts derived from Src-knockout mice showed unremarkable bone morphology with respect to wild-type animals (42)
or indicated a continuing increasing bone mass skeletal phenotype in Src–/– mice (43)
. Proteomic markers of osteoblast differentiation (24)
were also found unaltered in SI-83-treated and untreated primary osteoblasts. Cellular adhesion, regulated by Src activity in fibroblasts (53)
, was not affected by drug treatment in primary osteoblasts (data not shown). The therapeutic application of Src inhibitors may be of great relevance in bone diseases, since the compensation for the loss of Src activity by other members of the Src kinase family in other cell types probably does not occur in bone cells (44)
. Therefore, inhibition of Src would be predicted to affect bone without altering the metabolism of other organs (20)
.
In primary human osteoblasts SI-83 pyrazolo[3,4-d]pyrimidine does not impair differentiation and mineralization
Bone matrix is produced and deposed by osteoblasts in bone tissue, and the formation of mineralization nodules is one of the most important markers of their differentiation. To evaluate the effect of pyrazolo-pyrimidine derivatives on osteoblast biological functionality, after cell treatment, the deposed Ca2+ of the mineralized nodules was quantified, with PP2 and SI-83 administered to cells twice a week, in correspondence of medium changes. We used concentrations corresponding, respectively, to IC5, IC20, and IC50, previously calculated in SaOS-2. The treatments were carried out for 3 wk, and deposition of Ca2+ was measured each week (Fig. 5D
). Cells treated with PP2 did not tolerate drug treatment and started to die and to detach. Moreover, the percentage of Ca2+ present inside calcification nodules was always lower than control. This is probably due to the toxicity of PP2 in osteoblasts. On the contrary, the treatment with SI-83 was better tolerated since only at the highest concentration used and at the longest time could we see a worse nodule mineralization (Fig. 5D
).
The increased levels of calcium deposition and nodule formation we observed were in line with already reported data indicating that targeted disruption of Src-gene in a mouse model enhanced osteoblast differentiation and bone formation (45)
. This implies that Src plays an important role in osteoblast proliferation and differentiation, whereas it does not appear to modulate cell survival in these cells, since the down-regulation of Src activity is a well-established feature of differentiated osteoblasts (45
, 46
, 21)
. Moreover, alkaline phosphatase activity, evaluated in SI-83-treated primary human osteoblasts (Fig. 5E
), showed a similar trend as observed in the mineralization study (Fig. 5D
). Our results obtained in human osteoblasts after treatment with SI-83 confirmed previous results and indicated that our novel compound may be an interesting and new chemotherapeutic agent for bone sarcomas. Based on our data and on the above reported knowledge (18)
, Src could thus be the target of pharmacological treatments of diseased bone cells with a good chance of tolerable effects on other organs. Unaltered activity of primary osteoblasts was also confirmed by the analysis of proteome markers of osteoblast differentiation (24
, 25)
that were similarly expressed in SI-83-treated and untreated cultures (data not shown).
SI-83 pyrazolo[3,4-d]pyrimidine inhibits cell viability in five human osteosarcoma cell lines
Besides its action on SaOS-2 cells, SI-83 also inhibited cell viability of four more human osteosarcoma cell lines (Fig. 6
A). In particular, MNNG cells seemed to be even more responsive than SaOS-2 to the activity of the compound. On the other hand, TE-85, U-2 OS, and MG63 were less susceptible to SI-83. The different response of these cell lines may be in agreement with their different phenotype. MNNG and TE-85 were originally derived from the same osteosarcoma patient (47)
, but the MNNG cell line is a further transformed subclone from TE-85 and has far more malignant features in terms of metastatic and tumorigenic potential than its original clone (48)
. MG-63 cells, derived from low-grade osteosarcoma, poorly express TEM7, an important gene associated with metastasis, that is, on the contrary, highly expressed in SaOS-2 and TE-85 cell lines (49)
and is the only osteosarcoma cell line not affected by the action of the proapoptotic dasatinib Src inhibitor (41)
. SaOS-2 cells have been reported to be much more tumorigenic and metastatic with respect to U-2 OS (50)
. Moreover, SaOS-2 cells may mimic undifferentiated osteoblasts, while MG-63 cells may represent a more differentiated stage (51)
. This has been also proven by proteomics, indicating that SaOS-2 express proteins typical of immature osteoblasts (24)
. Moreover, in human osteosarcoma lines the E promoter of ERalpha transcription is active, except in MG-63, while primary osteoblasts express low levels of ERalpha proteins (52)
. These findings are in agreement with our observation that when pyrazolo[3,4-d]pyrimidine derivatives are used in human mature osteoblasts they only modestly affect proliferation and apoptosis.
|
SI-83 pyrazolo[3,4-d]pyrimidine decreases osteosarcoma tumor mass in a xenograft mouse model
To evaluate the effect of SI-83 in a preclinical experimental model of osteosarcoma, we injected subcutaneous SaOS-2 cells together with Matrigel in nude mice. The SaOS-2 xenograft showed an appreciable growth in 100% of the inoculated mice with a volume doubling time of 15 days. The daily administration of SI-83 did not determine any appreciable sign of distress or loss of weight in mice (data not shown). We divided mice in three groups containing each four mice: mice receiving vehicle (control), mice receiving 50 mg/kg SI-83, and mice receiving 100 mg/kg SI-83. The administration of SI-83 determined at endpoint a reduction in xenograft growth with respect to control measured as weight reduction (Fig. 6B1
). At the dose of 100 mg/kg, SI-83 determined a significant reduction (P<0.01) in tumor weight of 40% with respect to control tumors. In addition, the administration of 50 mg/kg SI-83 determined a smaller (15%) but significant reduction (P<0.05) in tumor volume with respect to control (Fig. 6B1
). Representative tumors for each group are shown in Fig. 6B2
. Xenograft tissues were processed for histological evaluation of phospho-Src expression (Fig. 6B3
). Tumor cells in the control group expressed detectable levels of phospho-Src. On the contrary, when we analyzed the tumor tissues from the treated groups, we detected only a faint or null staining for the phospho-Src antigen (Fig. 6B3
).
In this study, we attempted to modulate growth arrest and induce apoptosis in human osteosarcoma cells by applying a new Src inhibitor, namely SI-83. We showed that the interruption of nonreceptor Src tyrosine kinase signaling by this compound effectively prevents cell viability, proliferation, and DNA synthesis in SaOS-2 cells in a dose-dependent manner. In addition, Src inhibition markedly increased cellular apoptosis. Thus, Src effects appear to involve both apoptosis and the cell cycle, being a potential target for modulating growth signals in human osteosarcoma. SI-83 pyrazolo[3,4-d]pyrimidine could provide clinical benefits as a new chemotherapeutic agent. The effects of this novel compound have been validated in five different osteosarcoma cell lines, and it seemed that more aggressive cell types are more sensitive to SI-83 activity. On the contrary, primary human osteoblasts were significantly less affected by the antiproliferative and proapoptotic activities of SI-83, probably due to a different role of Src in osteoblast cells than in other cell types.
These in vitro observations got also an in vivo confirmation of efficacy of the new pyrazolo[3,4-d]pyrimidine, since it showed to significantly reduce xenograft SaOS-2 tumor mass in a murine model without apparent toxicity for animals.
Thus, in human osteosarcoma, modulation of Src activity may be a potential therapeutic target of this new compound, as it has a low toxicity toward nonneoplastic counterpart cells.
|
ACKNOWLEDGMENTS |
|---|
Received for publication September 25, 2007. Accepted for publication November 29, 2007.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
u
a, M., Teti, A. (2000) Tyrosine kinase src inhibitors: potential therapeutic applications. Drug News Perspect. 13,169-175[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  T. Akiyama, C. R. Dass, and P. F.M. Choong Novel therapeutic strategy for osteosarcoma targeting osteoclast differentiation, bone-resorbing activity, and apoptosis pathway Mol. Cancer Ther., November 1, 2008; 7(11): 3461 - 3469. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
