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Original Studies |
Endocrine, Polypeptide, and Cancer Institute, Veterans Affairs Medical Center and Departments of Medicine (G.H., A.V.S., B.S., A.P.) and Urology (R.D.), Tulane University School of Medicine, New Orleans, Louisiana 70112; and Department of Laboratory Medicine and Pathology, Mayo Clinic, (D.G.B.), Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Dr. Andrew V. Schally, Veterans Affairs Medical Center, 1601 Perdido Street, New Orleans, Louisiana 70112-1262.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Demonstration of the antiproliferative action of somatostatin (SST) and the presence of its receptors (SSTR) on endocrine tumors led to the application of stable SST analogs for hormonal treatment of these malignancies (8, 9, 10). In addition, SST analogs found other applications as carriers of radioisotopes for visualization and targeted radiotherapy of SSTR-positive tumors (11, 12, 13). Recently, we successfully used SST analogs for targeted delivery of doxorubicin or its derivative 2-pyrrolinodoxorubicin to cancers that express SSTRs (14). Various findings suggest that these new approaches may have application in the therapy of prostate cancer. The presence of SSTRs has been demonstrated in human prostate cancer cells and in histological specimens of primary and metastatic lesions of prostate cancers by different detection methods (15, 16, 17, 18, 19, 20, 21, 22, 23). Numerous studies showed that octapeptide analogs of SST can inhibit the growth of experimental prostate cancers (18, 19, 24). The therapeutic value of SSTR-targeted radionuclide therapy and chemotherapy was also demonstrated in hormone-independent rat and human prostate cancer models (20, 25, 26). Because the SST analog RC-160 (vapreotide) was shown to induce a temporary stabilization of disease in some patients with disseminated hormone-independent prostate cancer who no longer respond to androgen deprivation therapy (8, 27), it is reasonable to assume that SST analogs and their radio- or cytotoxic derivatives might likewise find applications as adjuvant treatment after radical prostatectomy. However, the findings on binding of SST octapeptides in surgical specimens of prostate cancer are limited, incomplete, and conflicting (15, 21, 23). Moreover, there is no comprehensive information on octapeptide binding and SSTR expression pattern in a large cohort of patients treated with radical prostatectomy, especially with regard to tumor stage and grade. To address these issues, in the present study we characterized the incidence and properties of SSTRs in 80 patients with organ-confined and locally advanced prostate cancer treated with radical prostatectomy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SST analog RC-160 (Vapreotide, Octastatin) was supplied by Debiopharm (Lausanne, Switzerland). Radioisotope 125I-labeled sodium was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). All other peptides and chemicals, unless otherwise mentioned, were obtained from Sigma (St. Louis, MO), Bachem (Torrance, CA), R\|[amp ]\|D Systems (Minneapolis, MN), or California Peptide Research, Inc. (Napa, CA).
Tissue samples from patients
Specimens of primary human prostate cancers were obtained from
80 patients at the time of radical prostatectomy at Mayo Clinic
(Rochester, MN; 58 specimens of tumors) and the V.A. Medical Center
(New Orleans, LA; 22 specimens of tumors). The characteristics of the
80 study patients are shown in Table 1
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The local internal review boards approved the collection and use of
these specimens for the current study. All analyses were first
conducted to meet the primary clinical requirement for patient
management, and only residual tissue was used in this study. After
surgical removal, selected portions of the prostate tissues were
flash-frozen in liquid nitrogen and sent on dry ice, together with the
pathology reports, to the Endocrine, Polypeptide, and Cancer Institute
of the V.A. Medical Center in New Orleans, LA, where all of the
receptor analyses were performed. Histopathological examination of each
specimen was undertaken to confirm the presence of cancer with minimal
admixed nonmalignant tissue (<20%) before the receptor studies. All
cancer samples were primary tumors and not metastases. The unprocessed
specimens and their membrane fractions were stored at -80 C until
analyses of SST binding sites and molecular biology studies. The
samples from Mayo Clinic, received up to August 1997, were used only
for ligand binding studies, whereas recent specimens from the V.A in
New Orleans were subjected to both RRAs and molecular biology
studies.
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Preparation of membranes for receptor binding studies was performed as described previously, with some modifications (28, 29). Briefly, the samples were thawed and cleaned, and then the specimens were homogenized in 50 mmol/L Tris-HCl buffer (pH 7.4) and supplemented with protease inhibitors (0.25 mmol/L phenylmethylsulfonylfluoride, 2 µg/mL pepstatin A, and 0.4% (vol/vol) aprotinin) using an Ultra-Turrax tissue homogenizer (IKA Works, Wilmington, NC) on ice. The homogenate was centrifuged at 500 x g for 10 min at 4 C to remove nuclear debris and lipid layer. The supernatant containing the crude membrane fraction was ultracentrifuged (L8–80M, Beckman Coulter, Inc., Palo Alto, CA) twice at 70,000 x g for 50 min at 4 C after resuspending in fresh buffer. The final pellet was resuspended in homogenization buffer and stored at -80 C until assayed. Protein concentrations were determined by the method of Bradford (30), using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA).
Radioiodinated derivatives of RC-160 were prepared by the chloramine-T
method and purified by reverse phase high performance liquid
chromatography in our laboratory (29). SSTR binding assays were carried
out as previously reported (29) with some modifications using in
vitro ligand competition assays based on binding of
[125I]RC-160 as radioligand to tumor membrane
fractions. This radioligand was well characterized previously and
showed high affinity binding to SSTR2 and SSTR5 (24, 25, 29, 31, 32).
In brief, membrane homogenates containing 40–120 µg protein were
incubated in duplicate or triplicate with 40,000-70,000 cpm
[125I]RC-160 and increasing concentrations
(10-12-10-6
mol/L) of nonradioactive peptides as competitors in a total volume of
150 µL binding buffer. Binding reactions were performed in 12 x
75-mm borosilicate glass tubes for 2 h at room temperature. At the
end of the incubations, the reactions were terminated by adding 250
µL ice-cold assay buffer to the tubes, and the bound ligand was
separated from free ligand by centrifugation at 4,500 x
g for 10 min at 4 C. The pellet was washed twice with 500
µL ice-cold buffer, and the radioactivity in the pellet of each tube
was counted in a 
-counter (Micromedic Systems, Huntsville, AL).
Preliminary experiments were performed with membrane protein
concentrations ranging from 20–250 µg/tube to determine the minimal
amount of protein required to assess specific binding at a satisfactory
level. We found that accurate results can be obtained within a range of
30–180 µg membrane protein in an incubation volume of 150
µL.
RNA isolation and RT-PCR
Total RNA was isolated using the Micro RNA Isolation Kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. After precipitation, the RNA pellet was suspended in sterile diethylpyrocarbonate-treated water and quantified spectrophotometrically at 260 and 280 nm. First strand complementary DNA (cDNA) was reverse transcribed from total RNA with Moloney murine leukemia virus reverse transcriptase. Distilled water was used as a negative control. Initially, 3 µg RNA were incubated with 300 ng random primer in diethylpyrocarbonate-treated water in a total volume of 38 µL for 5 min at 65 C and then cooled slowly to room temperature. The RNA-primer complex was then incubated in 50 µL reaction mixtures containing 50 U Moloney murine leukemia virus reverse transcriptase, 40 U ribonuclease inhibitor, 1 mmol/L of each deoxyribonucleotide triphosphate, 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, and 3 mmol/L MgCl2 for 60 min at 37 C, followed by a 5-min incubation at 94 C. The same reaction mixture without reverse transcriptase was used as the negative control to exclude the contamination of genomic DNA. After RT, 5 µL of the reaction products were subjected to PCR amplification. PCR was performed in a final volume of 100 µL containing 16 mmol/L (NH4)2SO4, 67 mmol/L Tris-HCl (pH 8.8 at 25 C), 0.01% Tween-20, 800 µmol/L deoxynucleotide triphosphates (200 µmol/L each of dATP, dGTP, dCTP, and dTTP), 2 mmol/L MgCl2, 2.5 U Taq DNA polymerase, and 150 ng of each of the sense and antisense primers. All of the reagents used for RT-PCR were purchased from Stratagene, with the exception of Taq DNA polymerase (ISC:BioExpress, Kaysville, UT). For amplification from first strand cDNAs, gene-specific primers for hSSTR1 (sense, 5'-TATCTGCCTGTGCTACGTGC-3'; antisense, 5'-GATGACCGACAGCTGACTCA-3'), hSSTR2 (sense, 5'-ATGGACATGGCGGATGAGCCACT-3'; antisense, 5'-TACTGGTTTGGAGGTCTCCATTGA-3'), hSSTR5 (sense, 5'-CGTCTTCATCATCTACACGG-3'; antisense, 5'-GGCCAGGTTGACGATGTTGA-3'), and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; internal control) were used (33, 34, 35). Plasmids containing cDNA for hSSTR1, hSSTR2, and hSSTR5 were used as positive controls. PCR was performed in a DNA thermal cycler (model 2400, Perkin-Elmer Corp., Norwalk, CT). After denaturation at 94 C for 5 min, samples were subjected to 45 cycles comprised of 94 C for 1 min, 62 C for 1 min, and 72 C for 1.5 min for SSTR2; 40 cycles of 94 C for 30 s, 55 C for 30 s, and 72 C for 30 s for SSTR1 and SSTR5; or 25 cycles of 94 C for 1 min, 60 C for 1 min, and 72 C for 1 min for GAPDH, followed by a final extension at 72 C for 5 min. PCR products were separated electrophoretically on an 1.5% agarose gel and stained with ethidium bromide.
Data analysis
Specific ligand binding capacities and affinities were calculated by the Ligand-PC computerized curve-fitting program described by Munson and Rodbard (36). To determine the types of receptor binding, equilibrium dissociation constants (Kd values), and the maximal binding capacity of receptors (Bmax), SST octapeptide binding data were also analyzed by the Scatchard method (37). The Spearman rank order correlation test was used to evaluate the correlation coefficients, and Fisher’s exact test was performed to compare the incidence of SST octapeptide binding in various groups. P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of specific SST-binding sites and characteristics of
binding of radiolabeled SST analog RC-160 to membrane receptors on
human prostate adenocarcinomas were determined using ligand competition
assays. Fifty-two of the 80 tumor preparations examined (65%) showed
RC-160 binding. Analyses of the typical displacement of
[125I]RC-160 by the same unlabeled peptide
revealed that the one-site model provided the best fit, indicating the
presence of one class of high affinity SSTR in membranes of human
prostate cancer specimens. The computerized nonlinear curve fitting and
the Scatchard plot analyses of the SST binding data (Fig. 1A) in 52 receptor-positive tumor
specimens indicated that the single class of binding sites had a mean
Kd of 9.44 ± 0.53 nmol/L (range, 3.18–21
nmol/L) with a mean Bmax of 754.8 ± 56.7
fmol/mg membrane protein (range, 189-1880 fmol/mg protein). The
Kd was positively correlated with the receptor
density (Bmax; r = 0.57; P
< 0.001), indicating that higher affinity was associated with lower
receptor concentration. Biochemical parameters essential to establish
the identity of specific binding sites were also determined. Thus, the
binding of [125I]RC-160 was found to be
reversible, time and temperature dependent, and linear with protein
concentration in the human prostate specimens examined (data not
shown). The specificity of SST binding was demonstrated by competitive
binding experiments using several peptides structurally related or
unrelated to SST. The binding of radiolabeled RC-160 was completely
displaced by increasing concentrations
(10-12–10-6
mol/L) of SST-14, whereas none of the structurally and functionally
unrelated peptides tested, such as LHRH, human GHRH, epidermal growth
factor, [Tyr4]bombesin, and insulin-like growth
factor I inhibited the binding of [125I]RC-160
at concentrations as high as 1 µmol/L (Fig. 1B).
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To examine the mRNA expression of SSTR1, SSTR2, and SSTR5, we
performed RT-PCR analyses on the 22 samples of prostate cancer obtained
from New Orleans. The expected size of PCR products amplified with gene
specific primers was 217 bp for SSTR1, 1104 bp for SSTR2, and 222 bp
for SSTR5, respectively (Fig. 2). The
mRNA expression pattern of these three subtypes as well as receptor
binding characteristics and clinical and pathological data for prostate
cancer specimens from New Orleans are shown in Table 2. In 19 of 22 cancers examined (86%),
RT of RNA followed by PCR amplification with specific primers produced
a fragment of the expected size for SSTR1. PCR products for SSTR2 could
be detected in 3 of 22 prostate cancer specimens (14%), whereas 14
samples (64%) showed mRNA for SSTR5 (Table 2). Of the 22 tumor
specimens, 2 expressed mRNA for all 3 SSTR subtypes examined (cases 4
and 5); 5 showed only SSTR1 (cases 2, 8, 10, 14, and 18), and 1
displayed only SSTR5 (case 1; Table 2). Most samples expressed both
SSTR1 and SSTR5, and only 1 tumor (case 19) displayed SSTR1 and SSTR2
together (Table 2). Of the 22 prostate cancer specimens, 2 samples
displayed none of the 3 subtypes examined (cases 3 and 11).
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No corresponding PCR products were detected from negative controls,
indicating that PCR products were generated from cDNA and not from
genomic DNA (Fig. 2). PCR amplification with human GAPDH gene-specific
primers generated a single product of the expected size (207 bp) from
all samples, confirming that no RNA degradation occurred during the
preparation (Fig. 2).
Correlation of SSTR expression with clinical and pathological findings
Table 3 shows SSTR status in
relation to patient age, cancer grade (Gleason score), and pathological
stage in 80 human prostate cancers. In 52 samples that showed
RC-160-binding sites, the concentration of SSTR
(Bmax) did not change significantly in relation
to Gleason score (Fig. 3A) or tumor stage
(Fig. 3B). Similarly, no correlation with Gleason score was found for
receptor affinity (Kd). In a subgroup of patients
at high risk of recurrent cancer (stage pT3 and/or Gleason score of
8–10), the incidence of RC-160 binding was similar to that observed in
the low risk group (Fig. 4). There was
also no correlation between the patient’s age and SSTR incidence or
binding characteristics (Table 3).
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). The correlation of DNA ploidy as a new putative
prognostic factor with receptor findings was also investigated in 58
samples; 41 (71%) specimens showed diploidy, 13 (22%) showed
tetraploidy, and 4 (7%) showed aneuploidy, but no significant
correlation was found between ploidy and SSTR incidence or binding
characteristics. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The possible usefulness of SST analogs in prostate cancer is suggested by numerous experimental studies. SSTRs have been detected on the Dunning R-3327 AT-1 rat prostate model (24, 25), on human prostate cancer cell lines in vitro (15, 17), and on xenografts of PC-3 and DU-145 human androgen-independent prostate cancers grown in nude mice (18, 19, 26). The direct antiproliferative effects of SST or its analogs have been demonstrated in human prostatic cancer cell lines in vitro (42). Moreover, the growth of rat Dunning R-3327 AT-1 and human PC-3 and DU-145 prostate cancers was significantly inhibited by the SST analog RC-160 in vivo (18, 19, 24). In contrast to the detection of binding sites for octapeptide SST analogs in an experimental setting, Reubi et al. (21) reported the lack of octreotide binding to 19 specimens of primary prostate cancer using in vitro receptor autoradiography. The study of Reubi et al. (21) was at variance with a preliminary report from our laboratory on the presence of high affinity binding of octapeptides RC-160 and RC-121 in a limited number of biopsy specimens of human prostate cancer (16). In the present study we examined the binding of radiolabeled SST octapeptide RC-160 to crude membrane preparations of 80 cancer specimens obtained after radical prostatectomy. Because clinical and pathological characteristics of the present cohort are similar to those reported in other studies (4, 6), it is likely that our findings on SSTR status in organ-confined and locally advanced prostate cancer are representative. Our results indicate that 65% of primary human prostatic carcinomas possess high affinity octapeptide-preferring SSTRs, as shown by a mean Kd of 9.4 nmol/L. The Bmax of these receptors (754.8 fmol/mg membrane protein) showed slight intersubject variability, but the average density of SSTR was virtually the same regardless of tumor stage or grade.
The discrepancy between the present findings and a previous report (21) may be explained by the difference in the affinity of octreotide and RC-160 to SSTR subtypes as well as by the use of different detection methods. Both SST analogs have similar high affinity to SSTR2; however, RC-160 displays approximately 1 order of magnitude higher affinity toward SSTR5 (31). The difference in the detection methods and receptor binding affinity of SST analogs may also account for the higher frequency of SSTR expression among metastatic lesions than that required by Nilsson et al., using in vivo imaging with [111In]-labeled DTPA-D-[Phe1]-octreotide (22). These findings may indicate a higher heterogeneity of the SSTR status on prostate cancer metastases compared to primary tumors and a possible loss of SSTRs during cancer spread. In contrast to the native SST, both of the two analogs show only negligible binding to SSTR1 (31). To further investigate the expression of octapeptide-preferring SSTRs in human prostatic carcinoma, we determined the expression of mRNA for SSTR subtypes 1, 2, and 5 in 22 samples using RT-PCR. We were able to demonstrate that SSTR1 mRNA was widely distributed, being present in 86% of human prostate cancers. This is in accordance with the findings of other groups applying the RT-PCR technique and in situ hybridization (21, 23). The presence of SSTR2 mRNA was shown in only 3 of 22 (14%) prostate cancer specimens. A rare expression of SSTR2 in primary prostate cancer could be the reason for the failure to detect octreotide-binding sites in smaller series of specimens (21). In contrast to the low incidence of SSTR2, the expression of SSTR5 was found in 65% of samples. The presence of SSTR5 in primary prostate tumors is in line with the findings of Sinisi et al. (23). Because SSTR5 was shown to mediate the antiproliferative action of RC-160 (32, 43), a high incidence of this SSTR subtype may have direct therapeutic importance. In 1 of 16 samples (6%), neither SSTR2 nor SSTR5 was detected despite the binding of [125I]RC-160. It is likely that this low affinity binding may correspond to SSTR3 (31), the expression of which was not investigated. Because [125I]RC-160 binding assay is highly sensitive and specific for the expression of SSTR2 and/or SSTR5, it is reasonable to assume that the expression pattern of SSTR subtypes found in the subset of 22 prostate cancers is characteristic of the whole cohort investigated in the present study.
SST analogs and their radionuclide or cytotoxic derivatives appear to be of importance for the treatment of nonoperable prostate cancer. We were able to assess octapeptide binding in a group of 42 patients who are at high risk of cancer recurrence, i.e. have locally advanced (pT3) or high grade (Gleason score 8–10) disease. Interestingly, the incidence of SSTR in these patients was as high as that found in the whole population tested in the present study. This observation implies that SST analogs linked to radionuclides and cytotoxic analogs of SST that can be targeted to SSTRs may find application as an adjuvant treatment after radical prostatectomy. Moreover, clinically available octapeptide analogs, such as octreotide, vapreotide (RC-160), or lanreotide, particularly in long-acting depot preparations, could be used concomitantly with androgen deprivation as an adjuvant hormonal therapy. In addition to a direct antiproliferative action, SST analogs may interfere with the aberrant activation of androgen receptors by locally released growth factors (44), thereby preventing androgen-independent regrowth of the cancer. Our finding that the incidence of octapeptide binding in prostate cancer is not affected by the progression of the disease implies that high frequency of SSTR might also occur in patients with nonoperable disease. This hypothesis is supported by the findings of Nilsson and colleagues, who visualized metastases of the relapsed prostate cancer using [111In -DTPA-D-Phe1]octreotide (OctreoScan) (22), although the detection rate was lower than the incidence of SSTRs in primary tumors reported in the present study. Similarly, preliminary clinical results with vapreotide in patients with androgen-independent prostate cancer (27) support the idea of the possible use of octapeptide SST analogs for treatment of relapsed prostate cancer, although another SST analog somatuline demonstrated no evidence of oncological activity by PSA or radiographic tests (45). Our preclinical experience with targeted cytotoxic SST analog AN-238 in androgen-independent rat and human prostate cancers (25, 26) also suggests that patients with relapsed and metastatic disease could benefit from therapy with AN-238 or radionuclide SST analogs.
In conclusion, the present study demonstrates a high incidence of octapeptide-preferring SSTRs in organ-confined and locally advanced human prostate cancers. These findings suggest that octapeptide SST analogs and their radionuclide and cytotoxic derivatives could be considered for adjuvant treatment in patients with a high risk of recurrent cancer after radical prostatectomy. Because the incidence of SSTRs on prostate cancer appears to be unaffected by tumor progression, these new modalities of treatment may also find application in cases of nonoperable disease, particularly in patients with advanced, androgen-independent prostate cancer who have relapsed after androgen deprivation therapy.
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Acknowledgments |
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Footnotes |
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2 On leave from Department of Neuroendocrinology, Medical Center of
Postgraduate Education, Warsaw, Poland.
Received November 15, 1999.
Revised February 25, 2000.
Accepted April 1, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
)
or [D-Trp6]LH-RH (
). Other unrelated
peptides, such as epidermal growth factor,
[Tyr4]bombesin, IGF-I, and human GHRH, did not displace
the radioligand (data not shown). One hundred percent specific binding
is defined as the difference between binding in the absence and that in
the presence of 10-5 mol/L RC-160. Each
point represents mean of triplicate determinations.
