A novel somatostatin conjugate with a high affinity to all five somatostatin receptor subtypes

Authors


Abstract

BACKGROUND

Somatostatin receptors (SRS, five subtypes) are expressed in a variety of human tumors, including most tumors of neuroendocrine origin, breast tumors, certain brain tumors, renal cell tumors, lymphomas, and prostate cancer. Somatostatin (SMS) triggers cytostatic and cytotoxic effects and has a general inhibitory effect on secretion mediated through its interaction with SRS. That is the basis for its use in the treatment of SRS-positive tumors. Radiolabeled SMS analogs can also be used for systemic radiotherapy and for diagnostic investigations.

METHODS

Sms-14 was conjugated to a periodate-activated dextran70 (mean molecular weight, 70 kD) by reductive amination. The human tumor cell line LCC-18, from a neuroendocrine colonic tumor, was used for stable transfection with each SRS gene separately; transfection was achieved with the expression system TETon (Clontech, Palo Alto, CA). Clones were selected by culturing with G418 and hygromycin B, and positive clones were identified by reverse transcriptase–polymerase chain reaction and binding of iodine-125–labeled SMS-14. The binding affinity for each SRS subtype was then determined for the SMS-dextran conjugate (with SMS-14 used as a positive control).

RESULTS

Sms-dextran70 showed high affinity binding to all five receptor subtypes. The IC50 values were between 3 and 80 nM.

CONCLUSIONS

This conjugate has a long circulation half-life (i.e., ≈27 hours after subcutaneous administration in mice) and, with high SRS pan-affinity demonstrated in this study, it has potential in the therapy of SRS-positive tumors. Currently, the clinical significance of SMS-dextran70 is being explored in a clinical Phase I–II study of patients with hormone-refractory prostate cancer. The outcome of this study will be reported when it is available. Cancer 2002;94:1293–7. © 2002 American Cancer Society.

DOI 10.1002/cncr.10299

Somatostatin (SMS) was described in 1973 as a hypothalamic peptide that inhibits the secretion of immunoreactive pituitary growth hormone.1 Sms acts as an endogenous inhibitory regulator of the secretory and proliferative responses of its target cells. The effects are mediated when SMS binds to its membrane receptor (SRS), which has five subtypes, SRS1–5. The five SRS bind natural SMS, SMS-14, and SMS-28 with low nanomolar affinity.2 Natural SMS has a short in vivo half-life due to enzymatic degradation, so more long-acting SMS analogs have been developed.3, 4 Octreotide is the best known and most clinically established.5 However, octreotide and other short synthetic peptide analogs have affinity only to two SRS subtypes, SRS2 and SRS5.

Srs are expressed in a majority of human tumors, including most tumors of neuroendocrine origin, breast tumors, certain brain tumors, renal cell tumors, lymphomas, and prostate cancer.6 Octreotide is frequently used in the treatment of patients with secreting neuroendocrine tumors. The treatment effect is palliation of hormonal symptoms.7 There is an increasing interest in using SMS in cancer treatment due to its potentially antiproliferative and even apoptotic actions.8–11 However, little is known about the effective dose and dose frequency, partly because of limitations of existing analogs regarding their SRS subtype affinity and in vivo half-life.

This study investigates the binding affinity of glycosylated SMS-14 (an SMS-14–dextran conjugate) to each SRS subtype. The human tumor cell line LCC-18 was used for stable transfection with each SRS gene separately, using a commercial expression system.

Materials and Methods

Dextran PM70 was supplied by Pharmacia Amersham Biotech AB (Uppsala, Sweden). Sodium metaperiodate (Merck AG, Darmstadt, Germany) was used for dextran activation. Sodium cyanoborohydride (Chemicon, Stockholm, Sweden) was used for reductive amination. Taurin (Sigma-Aldrich Chemie, Steinheim, Germany) and somatostatin (Ferring, Kiel, Germany) were used for dextran conjugation. PD-10 disposable Sephadex G-25 columns were used for separation and purification (Pharmacia Amersham Biotech AB, Uppsala, Sweden).

Conjugate Synthesis

The conjugate was prepared essentially as described previously.12 Briefly, ≈20 mg of dextran and 6 mg sodium periodate were dissolved in 1 mL of 0.1 M sodium acetate buffer at pH 5.5, and the solution was incubated on a magnetic stirrer for ≈15 hours in the dark at room temperature. After incubation, the solution was purified on a PD-10 column equilibrated and eluted with 0.2 M sodium acetate buffer at pH 6.0. Twenty mg activated dextran in 2 mL of 0.2 M sodium acetate buffer at pH 6.0 was mixed with 3 mg SMS and ≈4mg of cyanoborohydride. The solution was incubated with gentle shaking for 4 hours in the dark at 5 °C. After 4 hours of incubation, 20 mg of taurine was added and the solution was incubated for another 3 hours. Finally, the mixture was purified on a PD-10 column. A sample of the eluted conjugate was measured in a spectrophotometer at 280 nm. The absorbance was noted and compared with a SMS standard curve, and the peptide concentration of the conjugate was determined.

Transfection and Binding Assay

Hygromycin B (Clontech, Palo Alto, CA) and genetecin G418-sulphate (Life Tech, Paisley, United Kingdom) were used for the selection of positive clones. The following were used in the receptor binding assay: 6 well plates (Greiner, Frickenhausen, Germany), bovine serum albumin (Sigma, Steinheim, Germany), Bacitracin and Pefabloc SC protease inhibitor (Roth, Karlsruhe, Germany), SMS-14 (Sigma, Steinheim, Germany), iodine-125-SMS (Amersham, Buckinghamshire, United Kingdom), and a BCA protein assay kit (Pierce, Rockford, IL).

Generation of LCC-18 Clones Stably Expressing the Five Different Somatostatin Receptor Subtypes

The LCC-18 cell line was a kind gift from Dr. K. Öberg (Uppsala University Hospital, Uppsala, Sweden). It was established by Lundqvist et al. in 1991 from a neuroendocrine colonic tumor.1 The LCC-18 cells were cultured in RPMI-1640 medium containing 11 mmol glucose supplemented with 5% (volume per volume, v/v) fetal calf serum (FCS), streptomycin (100 μ/g/mL), 100 U/mL penicillin, transferrin (20 mg/500 mL), 17-β-estradiol (1.09 ng/mL), insulin (1.33 μg/mL), hydrocortisone (3.6 ng/mL), and sodium selenite (3.86 ng/mL) at 37 °C with 5% CO2.

INR1-G9 hamster glucoganoma cells14 were cultured in RPMI-1640 medium containing 11 mmol glucose supplemented with 10% (v/v) FCS, 100 μ/mL streptomycin, and 100 U/mL penicillin at 37 °C with 5% CO2. INR1-G9 cells expressed endogenous somatostatin receptors and were used as a positive control in binding experiments. Chemicals were supplied as follows: RPMI-1640, FCS, penicillin, and streptomycin were from GIBCO BRL (Life Technologies, Eggenstein, Germany), all other supplements were from Sigma (Deisenhofen, Germany), with the exception of insulin. Insulin was from Hoechst (Frankfurt, Germany).

To test the endogenous expression of SRS in LCC-18 cells and to test successfully transfected LCC-18 clones, we used reverse transcriptase–polymerase chain reaction (RT-PCR) with SRS-specific primers and the informative restriction analysis, respectively, as previously described.15 In addition, binding studies were done with radioactive labeled somatostatin-14 as described below.

RNA Purification

RNA from cultured LLC-18 cells was isolated using the RNAClean protocol from AGS (Heidelberg, Germany). Briefly, 3 mL of RNAClean supplemented with 300 μL chloroform were directly added to the cells, after the culture medium was aspirated from a cell culture dish 10 cm in diameter, and incubated for a few minutes. After that, RNA preparation was continued as previously described.15

Reverse Transcriptase Polymerase Chain Reaction, Polymerase Chain Reaction, and Analytic Gel Electrophoresis

RT-PCR and analytic gel electrophoresis were performed as previously described.15 The expression plasmid pTRE was from Clontech. All plasmids containing the whole coding sequence of the five human somatostatin receptors were a kind gift from Dr. F. Raulf (Sandoz Pharma Ltd., Basel, Switzerland). The specific sequences were based on the accession numbers M81829 for SRS1, M81830 for SRS2, M96738 for SRS3, L07833 for SRS4, and D16827 for SRS5 (Data Bank EMBL, DKFZ, Heidelberg, Germany).

Plasmids comprising the whole coding sequence of the five human somatostatin receptors were used, namely, pRc/CMV-SRS1–5.

Using these plasmids, the coding regions (Table 1) of the five SRS types were amplified by specific polymerase chain reaction (PCR) primers to introduce an EcoRI-site on the 5′-end and an XbaI-site on the 3′-end, to facilitate cloning into the doxycyclin-inducible expression system pTRE (Clontech). In Table 1, the cloned regions of the somatostatin receptors and their locations within the five SRS genes used for amplification of the somatostatin receptor sequences are summarized.

Table 1. Cloned Regions and Gene Locations for Somatostatin Receptor Subtypes 1–5
Srs subtype/accession no.Cloned regionGene location
1/M8182942–129642–63, 1296–1276
2/M8183028–122028–49, 1220–1202
3/M9673846–137846–67, 1378–1355
4/L0783347–128547–67, 1285–1267
5/D168271–11881–23, 1188–1168

PCR for cloning the five somatostatin receptors was carried out as described above, with the following exceptions: For all primer pairs the reaction mix contained 5% v/v dimethyl sulfoxide. Annealing temperature was 63 °C. Only 20 cycles were done.

One μg of plasmid DNA provided templates. After amplification, PCR products were cut with the restriction enzymes EcoRI or XbaI (New England Biolabs, Beverly, MA) and separated by gel electrophoresis as described for the PCR products above. The bands were cut out and purified using the QIAEX Agarose gel extraction protocol (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The expression plasmid pTRE (Clontech) was also cut with these enzymes and purified in the same way. The SRS fragments were then ligated into the pTRE-vector overnight at 16 °C using the DNA ligation kit from Stratagene (La Jolla, CA) according to the manufacturer's protocol. The new vector constructs were then transformed to E.coli DH5alpha by standard methods. After identification of positive clones by PCR using the same primer pairs as described for the cloning protocol, a large-scale preparation of each of five different expression plasmids was done by standard methods.

Generation of LCC-18 Clones Expressing the SstrSomatostatin Receptor Subtype Genes

According to the manufacturer's protocol for the Teton system (Clontech), we first established LCC-18 clones that stably expressed the regulator plasmid pTeton. Next, we used such clones to generate the SRS-expressing LCC-18 clones. Both transfection steps were performed by using the Perfect Lipids pFX-3 transfection kit from Invitrogene (San Diego, CA) according to the manufacturer's protocol. Clones containing the stably transfected regulator plasmid cells were selected by G418 (400 μg/mL) and screened for a high pTeton activity and a low background after treatment with doxycyclin (2 μg/mL) as an inductor by the reportergen construct pTRE-LUC from Clontech, according to the manufacturer's protocol. One clone with high pTeton activity was then selected for stable transfection of the five different SRS genes in the pTRE system. Following the manufacturer's protocol, after transfection with the plasmids pTRE-SSTR-X and pTK-Hyg (Clontech), positive clones were selected by hygromycin B (200 μg/mL). Clones of the second transfection step, growing well in the presence of hygromycin B, were then evaluated for expression of the five different SRS by RT-PCR using the PCR primers as described in Table 1, together with informative restriction analysis and binding studies with 125I-labeled SMS-14.

Binding Assay

Displacement of 125I-SMS with increasing concentrations of SMS-dextran70 or SMS-14 was performed. A total of 1.5–2 million cells per well were dispensed into a 6-well plate (2 wells per concentration of ligand) and incubated overnight at 37 °C with 5% CO2 pressure. The medium was removed and the wells were incubated with 1 mL assay buffer (phosphate-buffered saline [PBS] containing 1% BSA, 0.1% Bacitracin, and 1 mmol Pefabloc), SMS-14 or SMS-dextran70, and ≈30,000 cpm of 125I-SMS (specific activity was 2000 Ci/mmol). The incubation was for for 30 minutes at 37 °C. After the incubation, the incubation solution was removed and 1 mL 0.1 N NaOH per well was added to dissolve and lyse the cells. Then 0.75 mL of the cell solution was placed in a gamma counter for 3 minutes. The protein concentration was determined for each sample using a BCA Protein Assay Kit (10 μL cell lysate were added to the wells of a 96-well plate and 200 μL BCA reagent was added and incubated for 30 minutes at 37 °C). Finally, the absorbance was measured in a spectrophotometer at 570 nm. The protein concentration and the radioactivity/mg protein could be determined.

Results

The molar ratio between the dextran glucose units and the activating agent, sodium periodate, was 1:0.25. With this activation degree, 1.22 μmol SMS per 0.28 μmol dextran was coupled, i.e., an average of ≈4 SMS per dextran chain. The yield was highly reproducible. The principal formula of SMS-dextran70 is illustrated in Figure 1.

Figure 1.

The principal formula of somatostatin-dextran70. The number 388 indicates the average number of glucose units of the dextran backbone. The transparent amino acids indicate the receptor binding site. On average, 4 somatostatin receptor subtypes were coupled to the dextran backbone.

The IC50 values for SMS-dextran70 were between 3 and 80 nM. The binding assay was repeated several times and a variation in IC50 figures between assay runs was seen on SRS subtypes 1 and 4 (IC50 for SRS1 3–30, nM; for SRS4, 40–80 nM). For SRS2, 3, and 5, the variation was relatively small (IC50 for SRS2, 3–9 nM; for SRS3, 3–5 nM; for SRS5, 3–5 nM. The corresponding typical octreotide values for SRS2 and SRS5 were ≈2nM and ≈22nM, respectively.16 The variations within an assay, i.e., between duplicates, were small for all SRS subtypes (relative variation < 10%). One experiment is illustrated in Figure 2.

Figure 2.

The relative binding affinity of SMS-dextran70 to somatostatin receptor subtypes 1–5 compared with SMS-14 (the result of one experiment, mean of duplicates, relative standard deviation < 10%).;11>

Discussion

The antiproliferative effects of SMS in solid tumors have been demonstrated in vitro as well as in vivo: in vitro on cultured cells derived from both endocrine and epithelial tumors, and in vivo on 7,12-dimethylbenzanthracene–induced or transplanted rat mammary carcinomas (to give one example).17, 18 The effects are cytostatic (growth arrest) and cytotoxic (apoptosis). The growth arrest is triggered when SMS binds to SRS1, 2, 4, and 5 while the apoptosis mechanism is through binding to SRS3. There is also an indirect growth inhibition effect through SMS binding to SRS present on nontumor cells, resulting in inhibition of the secretion of growth-promoting hormones, inhibition of angiogenesis, and modulation of immune function.19–21

Octreotide is clinically well established as a means of controlling hormonal symptoms in patients with carcinoid syndrome. However, there are not many reported results of clinical trials of octreotide or other octapeptides in advanced malignancy.22, 23 There may be several possible reasons for these scarce results (e.g., the tumor burden in advanced disease); however, three important factors—metabolic stability, the efficacious concentration, and SRS subtype affinity—should be discussed. Although the octapeptide analogs are longer-acting than SMS-14, their biologic half-life is < 2 hours. This may result in failure to reach adequate, efficacious blood concentrations. The SRS subtype affinity for existing SMS analogs are limited to SRS2 and SRS5. This limits the probability of growth arrest, which would lead to growth control, and excludes the signal transduction that would lead to apoptosis by binding to SRS3. SMSSms-dextran70 does not have these limitations. It has very high metabolic stability, and the blood half-life in mice after subcutaneous administration is ≈27 hours.24 This indicates that it is possible to reach and maintain high blood concentrations, possibly more efficacious than before. The results of this study show that SMS-dextran70 has nM affinity to all five SRS subtypes; it has pan-affinity, and consequently interaction with all SRS subtypes is possible. The variation in affinity seen in SRS1 and SRS4 between assay runs may indicate that the incubation time needs to be longer for SMS-dextran70. With a longer incubation time, the molecule may adapt better to the binding pockets of SRS1 and 4 (keeping in mind the size of SMS-dextran70).

In conclusion, SMS-dextran70 has unique properties, and its clinical relevance is currently being explored in a clinical Phase I–II study. The outcome of this study will be reported when it is available.

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