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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 6 2218-2224
Copyright © 2006 by The Endocrine Society

Selective Activation of Somatostatin Receptor Subtypes Differentially Modulates Secretion and Viability in Human Medullary Thyroid Carcinoma Primary Cultures: Potential Clinical Perspectives

Maria Chiara Zatelli1, Daniela Piccin1, Federico Tagliati, Arianna Bottoni, Andrea Luchin, Cristina Vignali, Angelo Margutti, Marta Bondanelli, Gian Carlo Pansini, Maria Rosa Pelizzo, Michael D. Culler and Ettore C. degli Uberti

Section of Endocrinology (M.C.Z., D.P., F.T., A.B., A.L., C.V., A.M., M.B., E.C.d.U.), Department of Biomedical Sciences and Advanced Therapies, and Section of General Surgery (G.C.P.), Department of Surgery, University of Ferrara, 44100 Ferrara, Italy; Department of Medical and Surgical Science (M.R.P.), General Surgery III, University of Padua, 35100 Padua, Italy; and IPSEN Group (M.D.C.), Milford, Massachusetts 01757

Address all correspondence and requests for reprints to: Ettore C. degli Uberti, Section of Endocrinology, Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, Via Savonarola 9, 44100 Ferrara, Italy. E-mail: ti8{at}unife.it.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Medullary thyroid carcinoma (MTC) is a rare tumor originating from thyroid parafollicular C cells. We previously demonstrated that somatostatin (SRIH) reduces cell growth in the human MTC cell line, TT, which expresses all SRIH receptor (SSTR) subtypes and responds differently to selective SSTR agonists.

Objective: To clarify the possible effects of SRIH analogs on hormone secretion and proliferation in MTC primary cultures, we evaluated SSTR expression and assessed the in vitro effects on calcitonin (CT) and chromogranin A secretion as well as cell viability of SRIH analogs interacting with SSTR1, SSTR2, and SSTR5.

Design: Thirty-five patients affected by MTC were recruited from 2003 to 2005. After total thyroidectomy, the samples were examined for CT, chromogranin A, and SSTR expression by RT-PCR. Primary cultures were developed and tested with SRIH analogs interacting with SSTR1, SSTR2, and SSTR5.

Results: We selected 18 MTC tumor samples, expressing SSTR1, SSTR2, and SSTR5. Two different groups were identified according to CT secretion inhibition by the clinically available SRIH analog, lanreotide. In the responder group, CT secretion was reduced by compounds interacting with SSTR1, SSTR2, and SSTR5, whereas cell viability was not affected. On the other hand, in the nonresponder group, CT secretion was reduced by the SSTR1 selective agonist, whereas cell viability was inhibited by SSTR2 selective agonists.

Conclusions: Our data suggest that SRIH analogs might be useful in medical therapy of MTC because they could have antiproliferative effects despite the lack of antisecretory activity and vice versa.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
MEDULLARY THYROID CARCINOMA (MTC), an aggressive calcitonin- (CT) secreting tumor arises from thyroid parafollicular C cells. MTC can also occur as part of the multiple endocrine neoplasia type 2 (MEN 2) syndromes and familial MTC (1), representing 5–10% of all thyroid cancers (2). The most effective treatment for MTC, to date, is total thyroidectomy with central neck lymph node dissection (3), but persistent or recurrent disease is reported in 29–85% of cases (4, 5). Radiotherapy and chemotherapy are of limited value in advanced MTC (3); therefore, new therapeutic strategies are needed.

Somatostatin (SRIH) is a peptide with widespread distribution that inhibits hormone secretion and cell proliferation in normal and neoplastic human tissues by interacting with five distinct receptors (SSTRs) (6, 7). There is evidence for a highly variable SSTR expression pattern in MTC (8, 9), in which treatment with octreotide or lanreotide (Lan), clinically available SRIH analogs with high affinity for SSTR2 and SSTR5, may control symptoms but does not affect tumor size and patient survival rate (10, 11). We previously demonstrated that in the human MTC cell line, TT, which expresses all SSTR subtypes, SRIH, and selective SSTR agonists affect cell proliferation and CT secretion differently (12, 13). Therefore, distinct SSTR subtypes may differently influence cell growth rate and hormone secretion.

To clarify the possible effects of SRIH analogs on hormone secretion and proliferation in human MTC, we evaluated SSTR expression pattern in human MTC samples derived from 35 patients who underwent total thyroidectomy. We then selected 18 tumors expressing SSTR1, SSTR2, and SSTR5 subtypes and tested the effects of SRIH analogs selectively interacting with SSTR1, SSTR2, and SSTR5 on CT and chromogranin A (CgA) secretion and on cell viability in primary culture.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients

The samples derived from 18 MTC patients, selected on the basis of CT, CgA, SSTR1, SSTR2, and SSTR5 mRNA expression by RT-PCR analysis among 36 samples deriving from 35 patients diagnosed and operated on for MTC between 2003 and 2005 at the Sections of Endocrinology and at the Section of General Surgery of the University of Ferrara and at the Department of General Surgery III of the University of Padova. Patient 2 underwent two surgical procedures: total thyroidectomy first and then metastatic lymph node removal after 6 months.

Table 1Go shows patients’ characteristics and preoperative hormonal values. All patients (eight males and 10 females; aged 50.3 ± 4.6 yr) had undergone total thyroidectomy with central neck lymph node clearance and had histological and immunohistochemical diagnosis of MTC. Carcinoembryonic antigen (CEA) plasma levels were highly variable (5–800 ng/ml). The examined 18 samples included 13 thyroid samples and five metastatic lymph node samples, deriving from different patients.


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  		TABLE 1. Patients’ clinical data

 
Cell cultures

Tissue samples were collected in accordance with the guidelines of the local committee on human research, obtained under sterile conditions, and immediately minced in RPMI 1640 medium. Tissues were incubated with 0.25% trypsin overnight at 4 C and dissociated using 0.35% collagenase (Sigma, Milano, Italy) and 1% trypsin at 37 C for 60 min. Cell suspensions were filtered through double layers of gauze and washed twice with serum-free F-12 Ham’s nutrient-modified medium (F-12) (Euroclone Ltd., Wetherby, UK). Tumor cells were resuspended in F-12 with 10% fetal bovine serum and antibiotics, seeded in 96-well culture plates (~2 x 104 cells/well) and incubated at 37 C in a humidified atmosphere of 5% CO2-95% air, as previously described (14). After approximately 18 h, cells were treated with test substances, with further evaluation of hormone secretion and cell viability. Informed consent of the patients was obtained for disclosing clinical investigation and performing the in vitro study.

Isolation of RNA and RT-PCR

RT-PCR analysis for CT expression was performed on each specimen, and expression of CgA, SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 was analyzed only in CT-expressing tissues, as previously described (15). Briefly, total RNA was isolated from tissues with TRIzol reagent (Invitrogen, Milano, Italy) and treated with RNase-free deoxyribonuclease (Promega, Milano, Italy). Using the SuperScript preamplification system for first-strand cDNA synthesis (Invitrogen), 1 µg total RNA was reverse transcribed with random hexamers in the GeneAmp 9700 PCR system (Applera, Monza, Italy), as previously described (16). The cDNA (1 µl of RT reaction) was amplified by PCR with 1 U Taq DNA polymerase (Invitrogen). Integrity of cDNA was tested by demonstrating the presence of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal. PCRs were carried on using the oligonucleotide primers previously described (17, 18, 19). Each PCR product was digested by restriction enzyme and analyzed on 2% agarose gel to confirm the correct identification of the amplicons (data not shown).

Determination of endogenous control genes

The best endogenous control gene was determined before measurement of SSTR mRNA levels using the human endogenous control plate (Applera Italia) following the manufacturer’s instructions. Total RNA was isolated from three MTC samples and from a pool of three samples of normal thyroid tissue deriving from the same patient (calibrator). The candidate genes included 18S rRNA, acidic ribosomal protein, ß-actin, cyclophilin, GAPDH, phosphoglycerokinase, ß2-microglobulin, human ß-glucuronidase (huGUS), hypoxanthine ribosyl transferase, transcription factor IID TATA-binding protein, and transferrin receptor. All measurements were performed in duplicate. The results are expressed as cycle threshold values ({Delta}CT), representing the difference between CT obtained from the calibrator (normal thyroid pool) and the CT recorded for the samples for each housekeeping gene.

Quantitative PCR (QPCR) for SSTR1, SSTR2, and SSTR5 mRNA

QPCR for SSTR1, SSTR2, and SSTR5 was performed as previously described (14, 19). To assess retrotranscription efficiency, the predeveloped TaqMan assay reagents for huGUS were used (Applera Italia). All QPCRs were performed, recorded, and analyzed using the ABI 7700 Prism sequence detection system (Applera Italia). Slopes for all assays were –3.3 ± 0.1. No template control and RT controls were run in each experiment. A cut-off of 3 x 103 mRNA copies/µg total RNA was established as the threshold for QPCR to exclude the detection of transcripts due to illegitimate transcription (20, 21).

SRIH and SSTR selective agonists

Respective affinities to the different SSTRs of the used SRIH analogs, provided by Biomeasure Inc., IPSEN Group (Milford, MA), have been previously described (15, 22).

Cell viability

Cell viability was assessed by the cell proliferation kit I dimethylthiazoldiphenyltetrazolium bromide (Roche Diagnostics GmbH, Mannheim, Germany) in primary cultured cells plated in 96-multiwell plates (2 x 104 cells/well) and incubated for 48 h in a medium supplemented with 10% fetal bovine serum in the presence or absence of each SRIH analog at 10–8 M. Given the limited amount of primary cultured tissue, it was not possible to perform dose-response studies. Therefore, the 10–8 M dose was selected on the basis of compound affinity for each specific receptor and previous experience (14). Treatments were renewed after the first 24 h of incubation. The absorbance at 560 nm was recorded using a microplate reader (Victor; PerkinElmer, Monza, Italy). Results were obtained by determining the mean value of at least six experiments in eight replicates, as previously described (13).

CT assay

CT was measured in conditioned medium from primary cultured cells with the CT-U.S.-immunoradiometric assay kit (Biosurce Europe, Nivelles, Belgium), after a 6-h treatment without or with 10–8 M SRIH analogs. The intra- and interassay variation coefficients were 1.9–2.7 and 1.9–3.3%, respectively. The detection limit was 0.8 pg/ml. Assays were performed in duplicate. Results were obtained by determining the mean value among eight replicates. Primary cultures were considered as responders when a significant reduction greater than 15% in CT secretion was recorded under treatment with Lan.

CgA secretion

CgA secretion was analyzed by measuring human CgA immunoreactivity in the culture medium from primary cultured cells incubated for 6 h with or without 10–8 M each SRIH analog, with an ELISA kit (Dako, Milano, Italy), as previously described (16). The detection limit was 2.0 U/liter, with intra- and interassay coefficients of variation of 5.8 and 8.6%, respectively. Assays were performed in duplicate. Results were obtained by determining the mean value among eight replicates.

Statistical analysis

Data are expressed as the mean ± SE. A preliminary analysis was carried out to determine whether the data sets conformed to a normal distribution, and a computation of homogeneity of variance was performed using Bartlett’s test. The results were compared within each group and between groups using ANOVA. If the F values were significant (P < 0.05), Student’s paired or unpaired t test was used to evaluate individual differences between means. To measure the strength of association between pairs of variables without specifying dependencies, Spearman order correlations were run. P < 0.05 was considered significant in all tests.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
SSTR expression pattern in MTC

Thirty-six samples deriving from patients operated on for MTC underwent RT-PCR analysis, and 18 tumors (50%) were selected on the basis of the expression of CT, CgA, SSTR1, SSTR2, and SSTR5 mRNAs (Fig. 1Go and Table 2Go). The absolute SSTR mRNA levels were also investigated in the selected samples. We found that the best endogenous control gene was the huGUS (Fig. 2Go). {Delta}CT values for huGUS were little different from zero in all examined samples, indicating a stable gene expression level in the samples, compared with the calibrator. QPCR showed SSTR1 mRNA levels of 6.8 ± 2.2 x 105 copies/µg total RNA, SSTR2 of 7.3 ± 1.7 x 107 molecules/µg total RNA, and SSTR5 of 8.5 ± 4.9 x 104 copies/µg total RNA. SSTR mRNA expression levels are displayed in Tables 3Go and 4Go. SSTR2 mRNA expression levels were significantly (P < 0.01) higher than both SSTR1 and SSTR5 mRNA expression levels.


Figure 1
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  		FIG. 1. SSTR expression in MTC. RT-PCR was performed in all 36 MTC samples, showing the presence or absence of the expression of each SSTR. The bars indicate the percentage of positive samples.

 

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  		TABLE 2. Primary cultures in vitrohormone secretion and SSTR expression pattern

 

Figure 2
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  		FIG. 2. Gene expression profile of candidate endogenous control genes in MTC. Eleven genes were examined in three MTC by QPCR. IPC, Internal positive control; 18S, 18S rRNA; huPO, human acidic ribosomal protein; huBA, human ß-actin; huCYC, human cyclophilin; huGAPDH, human GAPDH; huPGK, human phospho-glycerokinase; huB2m, human ß2-microglobulin; huHPRT, human hypoxanthine ribosyl transferase; huTBP, human transcription factor IID TATA-binding protein; huTfR, human transferrin receptor. The results are expressed as {Delta}CT, representing the difference between CT obtained from the calibrator and the CT recorded for the samples for each housekeeping gene. The calibrator serves as a baseline for the assays and is considered as zero.

 

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  		TABLE 3. SSTR mRNA expression levels and percent CT secretion inhibition in the selected 18 MTC primary cultures undergoing treatment with 10–8 M SRIH analogs

 

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  		TABLE 4. SSTR mRNA expression levels and percent cell viability reduction in the selected 18 MTC primary cultures undergoing treatment with 10–8 M SRIH analogs

 
Effects of SRIH analogs on CT and CgA secretion by MTC primary cultures

To investigate the effects of SRIH analogs on hormone secretion, CT and CgA levels were measured in conditioned media collected from primary cultures derived from the selected 18 MTC. The primary cultures were then divided into two groups according to the extent of CT secretion inhibition recorded after treatment with BIM-23014 (Lan). Cultures responding to Lan with a CT reduction of 15% or greater vs. untreated cells were considered as responders. According to this criterion, nine cultures were considered as responders and nine as nonresponders. SSTR mRNA levels did not significantly differ between the two groups. Group A displayed SSTR1 mRNA levels of 9.1 ± 0.4 x 105 copies/µg total RNA, SSTR2 of 9.7 ± 0.3 x 107 copies/µg total RNA, and SSTR5 of 1.5 ± 0.1 x 105 copies/µg total RNA. Group B displayed SSTR1 mRNA levels of 4.6 ± 0.2 x 105 copies/µg total RNA, SSTR2 of 4.9 ± 0.1 x 107 copies/µg total RNA, and SSTR5 of 1.7 ± 0.03 x 104 copies/µg total RNA. Mean preoperative CT plasma levels were significantly lower in patients belonging to group A than in patients belonging to group B (158 ± 105 vs. 2383 ± 963 pg/ml; P < 0.01), whereas CEA plasma levels did not significantly differ between the two groups (402.5 ± 91.7 vs. 235.5 ± 33.1 ng/ml).

In group A, CT secretion was reduced by 27% after treatment with Lan (Fig. 3Go), and SSTR2 expression levels directly correlated with Lan inhibitory effects on CT secretion (r2 = 0.88; P < 0.05). Treatment with the SSTR1 selective agonist, BIM-23926, determined a significant reduction in CT secretion (–17%; P < 0.05), without correlation with SSTR1 expression levels. The inhibitory effect of the SSTR2 selective agonist, BIM-23120, on CT secretion (–23%; P < 0.05) directly correlated with SSTR2 expression levels (r2 = 0.5; P < 0.05). Treatment with the SSTR5 selective agonist, BIM-23206, inhibited CT secretion by 28% (P < 0.05), without correlation with SSTR5 expression. The dual SSTR2/SSTR5 agonist, BIM-23244, significantly reduced CT secretion (–25%; P < 0.05), and this effect directly correlated with SSTR2 expression (r2 = 0.7; P < 0.05). Finally, treatment with both the SSTR1 and the SSTR2 selective agonists (BIM-23926 + BIM-23120) had an additive effect (–38% CT secretion; P < 0.01), which directly correlated with SSTR2 expression levels (r2 = 0.67; P < 0.05).


Figure 3
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  		FIG. 3. Effect of SSTR agonists on CT secretion by MTC primary cultures. Primary cultured cells from 18 selected MTC samples were incubated in 96-well plates for 6 h with each SSTR agonist at 10–8 M. Control cells were treated with vehicle solution. CT levels in the conditioned medium were measured by immunoradiometric assay among eight replicates in the conditioned medium from treated and untreated (control) primary cultured cells. As described in Patients and Methods, samples were divided according to CT secretion inhibition after treatment with Lan in groups A (responders, black bars) and B (nonresponders, white bars). Data are expressed as the mean ± SE percent hormone secretion inhibition vs. untreated control cells. *, P < 0.05 vs. control.

 
In group B, not responding to Lan, we found that CT secretion was not reduced by treatment with SRIH analogs interacting with SSTR2, SSTR5, or both (BIM-23120, BIM-23206, BIM-23244). However, the SSTR1 selective agonist, BIM-23926, significantly reduced CT secretion (–13%; P < 0.05) (Fig. 3Go), without correlation with SSTR1 expression levels.

Concerning CgA secretion, SRIH analogs did not significantly affect CgA secretion in MTC primary cultures, in both groups A and B, except for MTC no. 8. In this sample, belonging to the nonresponder group and expressing all SSTRs, CgA secretion was significantly reduced after treatment with each SSTR selective agonist alone or in combination (ranging from –35% to –67% CgA secretion vs. control; P < 0.05) (data not shown).

Effects of SRIH analogs on MTC primary culture cell viability

To investigate the effects of SRIH analogs on MTC cell viability, dimethylthiazoldiphenyltetrazolium bromide assay was performed in 18 selected MTC primary cultures. In group A cell viability was not affected by treatment with any SRIH analog (Fig. 4Go). On the other hand, in group B, not responding to Lan in terms of CT secretion inhibition, cell viability was significantly reduced after treatment with Lan (–14%; P < 0.01), the SSTR2 selective agonist BIM-23120 (–13%; P < 0.05), or the dual SSTR2/SSTR5 selective agonist, BIM-23244 (–13%; P < 0.05). MTC cell viability was not affected by treatment with the SSTR1 selective agonist, BIM-23926, the SSTR5 selective agonist, BIM-23206, or the combination of the SSTR1 selective agonist and the SSTR2 selective agonist. No correlation was found between the inhibitory effects of SRIH analogs on cell viability and SSTR expression levels.


Figure 4
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  		FIG. 4. Effect of SSTR agonists on cell viability of MTC primary cultures. Primary cultured cells from 18 selected MTC samples were incubated in 96-well plates for 48 h with each SSTR agonist at 10–8 M. Control cells were treated with vehicle solution. Cell viability was measured by a colorimetric method among eight replicates from treated and untreated (control) primary cultured cells. As described in Patients and Methods, samples were divided according to CT secretion inhibition after treatment with Lan in groups A (responders, black bars) and B (nonresponders, white bars). Data are expressed as the mean ± SE percent cell viability reduction vs. untreated control cells. *, P < 0.05, and **, P < 0.01 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The ideal treatment for MTC is radical and early-stage surgery, which obtains biochemical cure in 75–90% of patients without lymph node involvement. However, biochemical cure is achieved in only 20–30% of patients with lymph node metastases and rarely in patients with more widespread disease (23). In patients with persistent disease, the challenge is finding an effective cure. SRIH analogs appear to have an impact on the symptoms and the production of CT in some MTC patients (3).

SSTR expression in MTC has been investigated both in vivo (24) and in vitro (8, 25, 26, 27), with differing results. Our data show that QPCR is highly sensitive to investigate gene expression and also to identify the appropriate housekeeping gene in MTC samples (28, 29). Ideally, the best control is expressed at a constant level in all samples, as we show for huGUS expression, indicating that this gene is a good candidate control, in agreement with previous reports (30).

To obtain results that could be useful from the clinical point of view, we subdivided the MTC samples into two groups, according to the in vitro response to the clinically available SRIH analog, Lan, in terms of CT secretion reduction. This selection criterion allowed us to identify two distinct patient groups because the group of responders had much lower in vivo CT plasma levels than the group of nonresponders. On the other hand, no difference was found between the two groups in terms of SSTR expression, CEA plasma levels, tumor node metastasis stage, or multiple endocrine neoplasia type 2 mutations. Our results show that group A responded to Lan and the other selective SSTR agonists in terms of CT secretion inhibition, indicating that functionally active SSTRs are indeed expressed by these primary cultures. This evidence indicates that the signaling pathway downstream from SSTRs regulating the secretory processes is intact in these samples. In addition, our data indicate an important role for SSTR2 in mediating the inhibitory effects of SRIH analogs on CT secretion because all the compounds interacting with this receptor efficiently suppressed in vitro CT secretion.

This hypothesis is further supported by the evidence that the extent of CT reduction induced by Lan and the SSTR2 selective agonist directly correlated with SSTR2 expression levels. On the other hand, no correlation was found between SSTR1 agonist inhibitory effects on CT secretion and SSTR1 expression levels, as reported previously (19), indicating that the inhibitory effects of SSTR1 selective agonists are independent of SSTR1 expression levels. Moreover, our results show that SSTR1 and SSTR2 agonists had an additive effect in reducing CT secretion in the responder group, suggesting the presence of a positive cross talk between these two receptors. Concerning the SSTR5 selective agonist, its inhibitory effects on CT secretion did not correlate with SSTR5 expression levels, as was also found in other experimental models (22).

These results suggest that the effects of SSTR1 and of SSTR5 selective agonists might also be influenced by other factors, independent of receptor expression. This hypothesis is further supported by the finding that the group of responders to SRIH analogs in terms of CT secretion did not respond in terms of cell viability reduction, reflecting the discrepancy between the in vivo inhibitory effects on CT secretion and the lack of a significant reduction in MTC tumor bulk under treatment with SRIH analogs (9, 31). Our data also confirm previous findings indicating a dissociation between the in vitro effects of SRIH analogs on tumor cell proliferation and hormone secretion (32). Taken together, these findings suggest that the signaling pathway downstream from SSTRs regulating cell proliferation is different from those modulating secretory activity.

The role for SSTR2 in transducing the inhibitory effects of SRIH on cell proliferation in MTC is further supported by the evidence that SRIH analogs interacting with SSTR2, but not with SSTR5, inhibited cell viability in group B. On the other hand, the SSTR1 selective agonist failed to affect cell viability, whereas it also significantly reduced CT secretion in nonresponder MTCs, partially confirming previously reported data (13). This evidence suggests that compounds interacting with this receptor might be useful for medical treatment of patients not responding to the clinically available SRIH analogs. Our data show that treatment with the SSTR1 selective agonist blocks the antiproliferative effects exerted by the SSTR2 selective agonist, indicating that the signaling pathways activated by these two receptors might interact. The molecular mechanisms underlying this evidence require further studies.

Taken together, our results, showing that the responder and nonresponder groups have the same SSTR expression pattern, would indicate that MTC response to SRIH analogs depends on not only SSTR expression levels but also other factors, such as other membrane receptors or intracellular mechanisms.

SRIH analogs failed to significantly affect CgA secretion, suggesting that the latter is not regulated by SSTR activation and that the inhibitory effects of SRIH analogs on CT secretion might not be mirrored by parallel changes in CgA secretion. This possibly indicates that evaluation of plasma CgA levels might not be useful to monitor the biochemical response to treatment of MTC with SRIH analogs, even if CgA plasma levels have been reported to correlate with basal plasma CT levels in MTC (33).

In conclusion, our results show that a selected group of MTC responds differently to SRIH analogs in vitro, providing a possible explanation for the relative failure of clinically available SRIH analogs in controlling MTC growth. Furthermore, our data indicate that performing primary cultures from human tissues is important to better clarify the effects of novel compounds on human cells. In addition, our results underline the need for further studies using new therapeutic tools interacting with molecular targets different from SSTRs, such as tyrosine kinase inhibitors (34, 35).


   Footnotes
 
This work was supported by grants from the Italian Ministry of University and Scientific and Technological Research (University of Ferrara: 60%–2005 and Ministero dell’Instuzione, dell’Università e dell Ricera 2005 060 839-004), IPSEN Fondazione Cassa di Risparmio di Ferrara, and Associazione Ferrarese dell’Ipertensione Arteriosa.

M.C.Z., D.P., F.T., A.B., A.L., C.V., A.M., M.B., G.C.P., M.R.P., and E.C.d.U. have nothing to declare. M.D.C. is employed by IPSEN Group.

First Published Online March 28, 2006

1 M.C.Z. and D.P. contributed equally to this study. Back

Abbreviations: CEA, Carcinoembryonic antigen; CgA, chromogranin A; CT, calcitonin; {Delta}CT, cycle threshold values; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; huGUS, human ß-glucuronidase; Lan, lanreotide; MTC, medullary thyroid carcinoma; QPCR, quantitative PCR; SRIH, somatostatin; SSTR, SRIH receptor.

Received February 14, 2006.

Accepted March 20, 2006.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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