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Keywords:

  • neuroendocrine tumors;
  • somatostatin;
  • antiproliferative agents;
  • lanreotide;
  • octreotide

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND.

Survival rates for gastrointestinal (GI) and bronchopulmonary (BP) neuroendocrine tumors (NETs) have not significantly altered (overall 67%, 5-year survival) in 30 years (1973-2004), whereas the incidence has increased (∼ 1000%) in the same time frame. No effective or specific antineoplastic agent is available for treatment, although somatostatin analogs inhibit tumor secretion. Given the coexistence of somatostatin and dopamine regulatory receptors on NET cells, the antiproliferative efficacy as well as the signaling and transcriptional targets of their ligands were evaluated.

METHODS.

The cytotoxic effects of 12 somatostatin/dopamine compounds were evaluated in 3 NET cell lines, and real-time polymerase chain reaction and enzyme-linked immunoadsorbent assay studies were performed to delineate antiproliferative signaling pathways.

RESULTS.

The atypical BP-NET, NCI-H720, was most sensitive to the sst5 analog BIM23206 (half-maximal concentration, 2.4 pM) and demonstrated similar sensitivity to lanreotide and the sst2 analog BIM23120. The typical BP-NET, NCI-H727, was most sensitive to BIM23120 (0.7 nM) and to the pan-somatostatin receptor analog (BIM23A779). The GI-NET, KRJ-I, was most sensitive to sst2,5 analogs lanreotide (1 nM) and BIM23244 (7.4 nM). Lanreotide activated extracellular signal regulated kinase-1/2 phosphorylation and p21WAF1/CIP1 transcription, but inhibited Ki-67 transcription. NCI-H720 was most sensitive to the sst2,5- and D2-selective compound BIM23A761 (4.2 nM), as was NCI-H727 (5.5 nM). KRJ-I did not respond to any chimeric analog. BIM23A761 activated c-Jun N-terminal kinase signaling and caused inhibition of Ki-67 transcription. P21WAF1/CIP1 transcription was activated only in NCI-H727 cells.

CONCLUSIONS.

The different responses of each individual cell line suggested that NETs from different locations arising from different neuroendocrine cells may require cell-specific antiproliferative agents based on the unique receptor profile of individual lesions. Cancer 2008. © 2008 American Cancer Society.

Neuroendocrine tumors (NETs)—previously considered “carcinoids”—account for 0.66% of all malignancies; however, it is noteworthy that the incidence has increased 3% to 10% per year over the last 30 years.1, 2 Although they predominantly occur in the gastrointestinal tract (67.5%), a substantial percentage is found in the bronchopulmonary system (25.3%).3 A paucity are evident in the ovaries (1.01%), testes (0.07%), or thymus (0.38%), but they can occur in any location that contains cells of the diffuse neuroendocrine system.1 In rare circumstances (<10%), such tumors may present as metastatic disease from an unknown primary site.4 Of note is the high proportion (22.4%) of associated noncarcinoid malignancies that occur concurrently with NETs, especially of the small bowel.1

It is also noteworthy that there has been no improvement in survival in the last 3 decades (Surveillance, Epidemiology, and End Results database 1973-2004),3, 5 and to our knowledge there exists no effective, safe, and targeted therapy for the disease. The overall 5-year survival rate for gastrointestinal NETs is 67.2%,5 and for bronchopulmonary NETs it is 50% to 88%,3 with a worse prognosis for atypical lesions that are defined by a higher mitotic count (2-10 mitoses per square millimeter in 10 high-power fields) and/or the presence of necrosis.6 Given the increasing incidence of these lesions and the failure to improve outcome, there is a substantial need to identify more effective treatment modalities.1, 5

Although the only curative therapy for NETs is surgery, >85% of NETs are metastatic at diagnosis, hence ab initio, <15% of patients have any likelihood of having curative intervention.7 Currently available therapy (biotherapy, radiotherapy, surgical resection, and debulking) focuses on disease stabilization and symptom control. Tumor regression is very rare and estimated at 2% to 5%.7 The majority of contemporary therapy for NET disease is based on empiric data derived from experience with adenocarcinomas; to our knowledge few adequately powered prospective studies exist, and it is unclear at a cellular and molecular level how the individual NETs studied differ from each other.5

The most thoroughly investigated group of agents are the long-acting somatostatin analogs, which include lanreotide and octreotide. These compounds, which have been assessed in phase 3 clinical studies,8 are effective in controlling most symptoms associated with the hypersecretory activity of NET cells. Lanreotide is effective in the treatment of active postoperative acromegaly,9 but the antiproliferative role of both lanreotide and octreotide in gastrointestinal and bronchopulmonary NETs is limited. Direct mechanisms of somatostatin analog action require the activation of 5 receptors (sst1-5), belonging to the family of G-protein coupled receptors, and the induction of cell cycle arrest or apoptosis, mainly through the regulation of phosphotyrosine phosphatase and mitogen-activated protein kinase activities.10 The indirect mechanisms involve inhibition of tumor angiogenesis and secretion of factors that are required for tumor growth.

Dopamine regulates secretion and gene expression mediated by D2 receptors coupled to Gαi proteins, with a resultant inhibition of adenylyl cyclase, decreased cyclic adenosine monophosphate production, and suppression of activated phosphokinase A.11 Recently, chimeric molecules that possess potent, selective agonist activity for both somatostatin and dopamine receptors have been synthesized. These compounds have been shown to be more effective in suppressing growth hormone and prolactin secretion from cultured human growth hormone–secreting pituitary adenomas than either octreotide or mono-receptor ligands alone.11-13 To our knowledge these agents have not been evaluated against human NETs of the gastrointestinal and bronchopulmonary system.

We analyzed the effects of 12 novel somatostatin analogs and somatostatin/dopamine chimeric compounds (Table 1) on 3 human NET (carcinoid) cell lines (an atypical bronchopulmonary NET: NCI-H720; a bronchial NET: NCI-H727; and a small intestinal NET: KRJ-I14, 15) to investigate whether different NETs require cell-specific antiproliferative agents based on the unique receptor profile of individual lesions. In addition, we examined the antiproliferative signaling mechanisms of somatostatin- and dopamine-mediated cell inhibition in each NET type.

Table 1. Human Somatostatin Receptor Subtype Specificity (IC50)39
Compoundsst1sst2sst3sst4sst5D2
  1. IC50 indicates half-maximal concentrations (nM); sst, somatostatin; D, dopamine; ND, not done.

Somatostatin-141.950.251.21.771.41ND
Somatostatin-281.860.311.35.40.4ND
BIM23023>10000.4286.88>10004.18ND
BIM239263.6>10001000833788ND
BIM2312010000.344121000213.5ND
BIM23206>10001661000>10002.4ND
BIM23244>10000.29133>10000.67ND
Lanreotide>10000.7598>100012.7ND
BIM23A7194015.610000.37ND
BIM23A7792.460.320.5720.50.56ND
BIM53061NDNDNDNDND114.7
BIM23A7584860.1324>1000278
BIM23A7606620.03160>10004215
BIM23A7616020.128196>10008.525

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Culture Conditions

KRJ-I and NCI-H720 cells were cultured as floating aggregates at 37°C with 5% carbon dioxide. KRJ-I cells were kept in Ham F12 medium (Gibco) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, Mo), penicillin (100 U/mL), and streptomycin (100 μg/mL).15-17 For NCI-H720, a 1:1 solution of Ham F12 and Dulbecco minimal essential medium was supplemented with final concentrations of FBS (5%), insulin (0.005 mg/mL), transferrin (0.01 mg/mL), sodium selenite (30 nM), hydrocortisone (10 nM), β-estradiol (10 nM), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid medium (10 mM), and L-glutamine (2 mM).14 The adhesive growing NCI-H727 cells were kept at 37°C in RPMI‒1640 medium containing final concentrations of FBS (10%), L-glutamine (2 mM), sodium pyruvate (1 mM), and glucose (2.5 g/L).14 For further processing, NCI-H727 cells were washed in phosphate-buffered saline (PBS) before trypsin–ethylenediamine tetraacetic acid was applied.

Proliferation Studies

After being spun for 5 minutes at 1500 × g, the pellets of the 3 cell lines were resuspended in each medium to a concentration of 5 × 105 cells/mL and seeded in 96-well plates at 100 μL with 5 × 104 cells/well (2 plates used per experimental condition). Lane 1 and 2 contained negative (medium only) and positive (pure cell suspension) controls.17 The BioMeasure (BIM) compounds were provided by IPSEN (Milford, Mass). Drugs were diluted in PBS and applied every 24 hours in final concentrations from 10−12 to 10−6.

Agent Profiles

The agent profiles and somatostatin and dopamine receptor subtype specificities (half-maximal concentration [IC50]) of each of the tested compounds are provided in Table 1. All compounds are stable in vitro and degradation is not an issue (unpublished data).

MTT Assay

After 72 hours of incubation (37°C; 5% carbon dioxide) with each compound, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to a final concentration of 0.5 mg/mL per well followed by additional incubation for 3 hours.17, 18 The reaction was stopped, and the formazan dye was solubilized by adding an equal volume (100 μL) of acid-isopropanol (0.01 N HCl in isopropanol). The optical density was read at 595 nanometers (nm) using a microplate reader (Bio-Rad 3500, Richmond, Calif).16, 17

Enzyme-Linked Immunoadsorbent Assay (CASE) Assay

The effects of somatostatin analogs, BIM23926 (sst1), BIM23120 (sst2), BIM23023, BIM23244 (both sst2,5), and BIM23206 (sst5), were measured on the extracellular signal regulated kinase-1 (ERK1/2) signaling pathway as described.19 In separate studies, the effects of lanreotide on ERK1/2 signaling pathway and BIM23A761 on ERK/protein kinase B (AKT)/c-Jun N-terminal kinase (JNK) and nuclear factor κB (NFκB) signaling pathways were evaluated.19 Cells were stimulated with IC50 concentrations (from proliferation studies, Table 2) for 30 minutes. To assess the specificity of ERK pathway activation, cells were preincubated with PD98059 (30 μM) (an inhibitor of ERK1/2 phosphorylation) for 10 minutes before addition of lanreotide and BIM23A761. ERK, AKT, JNK and NFκB phosphorylation were measured using an enzyme‒linked immunoadsorbent assay (ELISA) (CASE, SuperArray, Hamburg, Germany) as per the manufacturer's protocol. Briefly, stimulated cells were fixed (4% formaldehyde), and stained with primary antibodies against either the nonphosphorylated or phosphorylated forms of each protein (60 minutes, room temperature [RT]). After washing and secondary antibody application (60 minutes, RT), cells were incubated with color developer (10 minutes, RT), and plates were read at 450 nm. Thereafter, protein was assayed in each well (protein development reading at 595 nm). Results were calculated as antibody reading (at 450 nm)/protein concentration (measured at 595 nm) and normalized to unstimulated cells. Phosphorylated signal was compared with total nonphosphorylated signal.

Table 2. Summary of Effects of Somatostatin Analogs and Somatostatin/Dopamine Chimeric Compounds on Neuroendocrine Tumor Cell Line Proliferation and ERK1/2 Phosphorylation
CompoundActionH720ERKpH727ERKpKRJ-IERKp
  1. ERK indicates extracellular regulated kinase; sst, somatostatin.

BIM23023sst2/5 agonist0.23 NmYesNo effectNo0.28 μMYes
BIM23926sst1 agonistNo effectNoNo effectNo8.5 nMYes
BIM23120sst2 agonist41 pMYes0.7 nMYesNo effectNo
BIM23206sst5 agonist2.4 pMYesNo effectNoNo effectNo
BIM23244sst2/5 agonist0.11 nMYesNo effectNo7.4 nMYes
Lanreotidesst2/5 agonist6.5 pMYesNo effectNo1 nMYes
BIM23A719Pan sst agonist 16.2 nMNo effect0.53 nM
BIM23A779Pan sst agonist 2No effect1.9 pM0.35 μM
BIM53061Dopamine agonist21 nMNo effect0.71 μM
BIM23A758sst/da agonist 1No effectNo effectNo effect
BIM23A760sst/da agonist 218.3 nMNo effectNo effect
BIM23A761sst/da agonist 34.2 nMNo5.5 nMNoNo effectNo

Real-Time PCR

Ribonucleic acid (RNA) was extracted from 2 × 106 NCI-H70, NCI-H727, and KRJ-I cells in log phase growth (n = 3) or from normal brain (n = 3) (TRIZOL, Invitrogen, Carlsbad, Calif), and cleaned (Qiagen RNeasy kit and DNeasy Tissue kit; Qiagen Inc., Germantown, Md) to minimize contaminating genomic DNA. RNA (2 μg) was converted to cDNA (High Capacity cDNA Archive Kit; Applied Biosystems).17, 20, 21 Real-time reverse transcriptase polymerase chain reaction (PCR) analysis was performed using Assays-on-Demand products and the ABI 7900 Sequence Detection System according to the manufacturer's suggestions.17, 20, 21 Cycling was performed under standard conditions (TaqMan Universal PCR Master Mix Protocol), and data were normalized using geNorm22 and expression of the novel house-keeping genes, ALG9, TFCP2, and ZNF410.17, 21

Receptor Profile

Levels of sst1-5 and D1-5 were measured in NCI-H720, NCI-H727, and KRJ-I cell lines. Normal brain was used as a control. Receptor probes were used to quantify the presence of somatostatin and dopamine receptors in each cell line. Transcript levels of <0.1 normalized geNorm were considered absent.

Cell Cycle Markers

Ki-67 and p21WAF1/CIP1 transcripts were measured in response to lanreotide and BIM23A761. NCI-H720, NCI-H727, and KRJ-I cells were stimulated with IC50 concentrations of lanreotide and BIM23A761, respectively. Cells were cultured for 24 hours before RNA extraction. RNA was isolated using the standard PCR protocol.17, 20, 21

Statistical Evaluation

All statistical analyses were performed using Prism 4 (GraphPad Software, San Diego, Calif). Sigmoidal dose responses and nonlinear regression analyses were calculated to identify half-maximal concentrations (IC50) for each drug. Alterations in signal transduction and transcriptional activation were assessed using 2-tailed Student t test for paired data.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Receptor Profile by PCR

Analysis of somatostatin and dopamine receptor levels using real-time PCR in each cell line demonstrated a different expression profile for each cell line.

NCI-H720

This cell line was derived from an “atypical” bronchopulmonary NET.14 It is specific for somatostatin receptors 2 and 5 as well as the dopamine receptors 2 and 4 (Fig. 1). The somatostatin/dopamine receptor profile of NCI-H720 is thus designated as sst2,5/D2,4.

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Figure 1. Somatostatin (SST) and dopamine (DA) receptor profiles in NCI-H720 (atypical), NCI-H727 (typical), and KRJ-I were assessed by real-time polymerase chain reaction (PCR). sst1, sst3, and sst5 receptors were found exclusively in NCI-H727 and KRJ-I cell lines (A, C, and D); sst2 were present in all cell lines (B). D1, D2, D4, and D5 receptors were present in NCI-H727 cells (E and F), whereas inhibitory D2 and D4 receptors were identified in NCI-H720 (G). D5 receptors were observed in KRJ-I cells (H). All sst and D receptors were identified in brain tissue (positive control). Mean ± standard error of the mean (SEM); n = 3.

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NCI-H727

NCI-H727 is a cell line derived from a “typical” bronchial NET.14 It is positive for sst1-3,5 in addition to D1,2,5. Very low levels of D4 (<0.1 normalized geNorm) were also identified (Fig. 1). The receptor profile of NCI-H727 is therefore sst1,2,3,5/D1,2,5.

KRJ-I

KRJ-I is derived from a “typical” small intestinal NET.15 It is positive for sst1-3,5 as well as low levels of D5 (= 0.1 normalized geNorm) (Fig. 1). The receptor profile of KRJ-I is sst1,2,3,5/D5.

Antiproliferative Effects of Somatostatin and Somatostatin/Dopamine Chimeras

NCI-H720

Proliferation of this cell line is inhibited by the sst5 agonist, BIM23206 (IC50 = 2.4pM), with a comparable sensitivity to lanreotide (sst2,5 agonist, 6.5 pM) and the sst2 agonist, BIM23120 (IC50 = 41 pM) (Table 2). NCI-H720 cells were less responsive to the sst2,5 agonist, BIM23244 (IC50 = 0.11 nM) and did not significantly respond to the sst1 agonist, BIM23926. These results are consistent with the somatostatin receptor profile of the NCI-H720 cells (Figs. 1A, 1B, and 1D) and with ERK1/2 phosphorylation induced by these somatostatin analogs (Table 2). Of the 2 pan-somatostatin receptor agonists tested, NCI-H720 cells were most sensitive to BIM23A719 (pan-somatostatin receptor agonist 1, IC50 = 6.2 nM).

Cell proliferation was also inhibited by the dopamine agonist, BIM53061 (IC50 = 0.23 nM) and both BIM23A760 (sst/da agonist 2, IC50 = 18.3 nM) and BIM23A761 (sst/da agonist 3, IC50 = 4.2 nM).

NCI-H727

The typical BP-NET was most sensitive to the sst2 agonist, BIM23120 (IC50 = 0.7 nM) and to the pan-somatostatin agonist 2, BIM23A779 (IC50 = 1.9 pM) (Table 2). Neither sst1, sst2,5, nor any of the sst2, 5 agonists caused inhibition of proliferation despite expression of transcripts for these receptors (Figs. 1A, 1B, and 1D). These results are consistent with the absence of ERK1/2 phosphorylation by these agents (Table 2).

Cell proliferation was also inhibited by the sst2,5- and D2-selective compound BIM23A761 (IC50 = 5.5 nM).

KRJ-I

KRJ-I was most sensitive to lanreotide (IC50 = 1 nM), BIM23244 (sst2,5 agonist, IC50 = 7.4 nM), and BIM 23926 (sst1 agonist, IC50 = 8.5 nM), but was not responsive to either a pure sst2 (BIM23120) or sst5 (BIM23206) agonist (Table 2). The cell proliferation inhibitory effects were consistent with ERK1/2 phosphorylation by these compounds; neither BIM23120 nor BIM23206 induced phosphorylation (Table 2). KRJ-I cells were sensitive to both pan-somatostatin receptor agonists, but were most sensitive to the type 1 agent (BIM23A719).

KRJ-I cell proliferation could not be effectively inhibited by either the dopamine agonists (IC50 = 0.7 μM) or the sst/da agonist chimeras, which reflects the absence of significant transcripts for the inhibitory dopamine receptor (type 2) on this cell line (Fig. 1F).

Effects of Lanreotide and BIM23A761 on Cell Signaling Pathways

NCI-H720

Lanreotide activated mitogen-activated protein kinase (MAPK) signaling (182 ± 35%, P<0.01 vs control) through phosphorylation of ERK1/2, which could be inhibited (65 ± 17, P < .01 vs lanreotide) by the MAPK/ERK kinase (MEK) inhibitor, PD98059 (Fig. 2A). This is consistent with the inhibitory effects of lanreotide on NCI-H720 cell proliferation. When cells were stimulated with the type 3 sst/da chimera, BIM23A761, ERK was not phosphorylated. In contrast, the JNK pathway was activated by this compound; phosphorylated JNK levels were elevated (61 ± 4%, P < .05 vs control) (Fig. 3A). Neither NFκB nor AKT were significantly altered by BIM23A761.

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Figure 2. Effects of lanreotide on extracellular regulated kinase (ERK)-1/2 phosphorylation are shown. Lanreotide (LAN) phosphorylated ERK1/2 in NCI-H720 and KRJ-I cell lines (A and C). Lanreotide had no effect on ERK1/2 phosphorylation in NCI-H727 (B). The effects of lanreotide were inhibited by the MEK inhibitor, PD98059 (1 μM) (A-C). *P < .01 versus control (Con); **P < .05 versus control; #P < .05 versus lanreotide. Mean ± SEM; n = 4.

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Figure 3. Effects are shown of BIM23A761 on cell signaling pathways. BIM23A761 inhibited ERK1/2 phosphorylation and activated JNK signaling in NCI-H720 and NCI-H727 cells (A and B). BIM23A761 has no effect on signaling pathways in KRJ-I cells (C). *P < .05 versus control; #P < .05 versus control. Mean ± SEM; n = 4.

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NCI-H727

Lanreotide had no effect on ERK phosphorylation (Fig. 2B), which is consistent with the observation that proliferation could not be inhibited (Table 2). The BIM23A761 sst/da chimera was associated, as in NCI-H720 cells, with significant activation of JNK phosphorylation (475 ± 27%, P < .01 vs control; Fig. 3B). Although ERK1/2 signaling was inhibited (17 ± 3, P < .05 vs control), neither the NFκB nor AKT signaling pathways were significantly activated.

KRJ-I

Lanreotide activated ERK1/2 signaling by 25% ± 8% (P = .05 vs control), which could be inhibited by the MEK inhibitor, PD98059 (Fig. 2C). BIM23A761 did not significantly alter phosphorylation of any of the signaling pathways, which was consistent with the lack of any demonstrable effect of this sst/da chimera on KRJ-I cell proliferation (Table 2).

Effects of Lanreotide and BIM23A761 on Downstream Transcriptional Targets

NCI-H720

Real-time PCR analysis of cells incubated with lanreotide for 24 hours demonstrated that Ki-67 transcript levels were significantly decreased (45% ± 7%) compared with unstimulated cells (P < .01) (Fig. 4A). This is consistent with the observation that lanreotide inhibits NCI-H720 proliferation. The effect was specific, because it could be inhibited by PD98059 (6% ± 2%, P < .05 vs lanreotide alone). Lanreotide significantly increased p21WAF1/CIP1 transcript levels compared with unstimulated cells (140% ± 27%, P < .01 vs control), and this increase was reversible by PD98059 (P < .05 vs lanreotide alone) (Fig. 4D). These results are consistent with the antiproliferative effects of this compound on NCI-H720.

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Figure 4. Real-time polymerase chain reaction (PCR) analysis of the effect of Lanreotide on Ki-67 and p21WAF1/CIP1 transcripts is shown. Lanreotide significantly inhibited Ki-67 transcription in all cell lines (A-C). P21WAF1/CIP1 transcript was stimulated in all cell lines (D-F). These effects were inhibited by PD98059 (1 μM). *P < .01 versus control; #P < .05 versus lanreotide. Mean ± SEM; n = 4.

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Real-time PCR analysis of BIM23A761-stimulated cells demonstrated that Ki-67 transcript levels were decreased 99% ± 17% (P < .01 vs controls) (Fig. 5A), confirming that this compound inhibited NCI-H720 cell proliferation. In addition, and in contrast to the effects of lanreotide in these cells, p21WAF1/CIP1 transcript levels were significantly decreased (74% ± 27%, P < .05 vs control).

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Figure 5. Real-time polymerase chain reaction (PCR) analysis of the effects of BIM23A761 on Ki-67 and p21WAF1/CIP1 transcripts is shown. BIM23A761 significantly inhibited Ki-67 transcription in NCI-H720 and NCI-H727 cell lines (A and B). Transcripts for p21WAF1/CIP1 were inhibited by BIM23A761 in NCI-H720 (D) and stimulated in NCI-H727 cells (E). No significant effects were identified in KRJ-I cells (C, F). *P < .01 versus control; #P < .05 versus control. Mean ± SEM; n = 4.

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NCI-H727

Real-Time PCR analysis of the effect of lanreotide on Ki-67 demonstrated that this was not significantly decreased compared with control cells (Fig. 4A), and p21WAF1/CIP1 transcripts were not significantly up-regulated (Fig. 4E), results consistent with the absence of an inhibitory effect of lanreotide on NCI-H727 proliferation.

PCR assessment of BIM23A761 demonstrated significant (P < .01) down-regulation of Ki-67 transcript levels (80% ± 12% change vs control) (Fig. 5B), whereas p21WAF1/CIP1 transcripts were elevated (1082% ± 97%, P < .01 vs control).

KRJ-I

Real-time PCR measurements of Ki-67 transcript levels demonstrated that lanreotide down-regulated Ki-67 (73% ± 12%; P < .01 vs control) (Fig. 4C), and this was inhibited by PD98059 (P < .05 vs lanreotide alone). Transcripts for p21WAF1/CIP1 were significantly elevated (175% ± 21%, P < .01 vs control) (Fig. 4F), results consistent with the antiproliferative effects of this compound on KRJ-I.

In BIM23A761-treated KRJ-I cells, neither Ki-67 nor p21WAF1/CIP1 transcript levels were significantly decreased (Fig. 5C and 5F), consistent with observations that BIM23A761 did not inhibit KRJ-I cell proliferation.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

This study reveals that individual lung and gastrointestinal NET cell lines exhibit substantially different receptor expressions for somatostatin and dopamine receptors, which are differently coupled to the ERK and JNK signaling pathways. In addition, somatostatin analogs and somatostatin/dopamine chimeric compounds are potent inhibitors of NET cell proliferation, and cell lines from the same organ (lung, typical vs atypical NET) were differently sensitive to receptor targeting. These molecular observations confirm the need to individualize therapeutic strategies based upon analysis of the receptor profile of a specific lesion as opposed to a global strategy based purely on a histopathological characterization or classification. The differences in ERK signaling activation to specific somatostatin analogs suggest that defining the receptor profile alone may not be sufficient to predict NET responsiveness to these analogs. This is highlighted by the results with lanreotide, BIM23244, and BIM23023, which are all predicted to inhibit cell function through activation of sst2,5. In the current study, lanreotide was the most effective inhibitor in NCI-H720 and KRJ-I cells. In contrast, and despite expression of sst2,5, none of these compounds inhibited NCI-H727 cell proliferation. The absence of an effect in this cell line may suggest either abnormal receptor expression (cell membrane/protein level), as has been noted in acromegalic tumors,23 or receptor coupling resulting in nonfunctional and divergent signaling pathways, as noted in rat somatotrophs.24 Because ERK1/2 phosphorylation studies demonstrated no responses to sst2,5 ligands in NCI-H727 cells, but did demonstrate an effect in NCI-H720 and KRJ-I cells, we conclude that effective receptor signaling may be a prerequisite for appropriate inhibition of NET proliferation.

An examination of pan-somatostatin receptor agonists demonstrated that the type 1 (BIM23A719) was most effective in NCI-H720 and KRJ-I cells (IC50 in NCI-H720 = 6.2 nM and IC50 in KRJ-I = 0.53 nM), whereas the type 2 agonist (BIM23A779) was most effective in NCI-H727 cells (IC50 NCI H727 = 1.9 pM). Of note is the finding that the pan-somatostatin receptor agents were not as effective as individualized somatostatin agonists in the atypical bronchopulmonary NET (NCI-H720), as in the “typical” bronchopulmonary NET. The sensitivity of the latter to a pan-somatostatin compound conforms to a previous study demonstrating that SOM230 was more effective than octreotide in inhibiting NCI-H727 proliferation.25 This also confirms our observation that NCI-H727 cells are not effectively inhibited by an sst2,5 compound, despite expressing these receptors.25, 26

Targeting the D2 receptor is an effective mechanism for suppressing NET secretion,11-13 but its antiproliferative effects are not well characterized, particularly on lung and gastrointestinal NETs. Targeting D2, which is coupled to Gαi and through coupling to phosphodiesterase activity reduces intracellular cyclic adenosine monophosphate,27 is associated with inhibition of MAPK signaling.28 Activation of inhibitory D2 receptors would therefore be predicted to inhibit cell proliferation. In the current study, NCI-H720 cells that express the inhibitory dopamine receptors (D2,4R) exhibit a decrease in cell proliferation when exposed to BIM53061 (IC50 = 21 nM). This compound also inhibits KRJ-I (μM concentrations) but not NCI-H727 cell proliferation. These differences could reflect expression of the stimulatory D1,5 receptors in these cell lines, and suggests that a D2 agonist would most likely be ineffective in neoplastic neuroendocrine cells expressing multiple dopamine receptor subtypes.

Somatostatin/dopamine chimeras target multiple receptor complexes to effect growth inhibition.13 Proliferation of NCI-H720 was inhibited by both sst/da agonists 2 and 3 (IC50: 4.2-18 nM). These agents, however, were less effective (<100-fold) than targeting specific somatostatin receptors alone, eg, sst5 or the pan-somatostatin receptor agonist 1 on NCI-H720 cells. Targeting the dopamine in addition to somatostatin receptors is not therefore additionally inhibitory in this cell line. In NCI-H727 cells, sst/da agonist 3 was an effective inhibitor of proliferation. The KRJ-I cell line was not effectively inhibited by any of the sst/da compounds.

Having determined that NET cells lines were differentially sensitive to somatostatin analogs and sst/da chimeras, we next evaluated the mechanisms (signaling pathways, downstream transcriptional targets) by which 2 agents, lanreotide (sst2,5) and BIM23A761 (sst/da agonist 3), inhibited cell proliferation. These agents were chosen because they were both potent (lanreotide: 6.5 pM NCI-H720 and 1 nM KRJ-I; BIM23A761: 4.2 nM NCI-H720 and 5.5 nM NCI-H727) and they significantly inhibited only 2 (lanreotide: H720 and KRJ-I; BIM23A761: NCI-H720 and NCI-H727) of the 3 cell lines; 1 cell line therefore could be used as a negative control.

Lanreotide stimulated ERK1/2 activity in both NCI-H720 and KRJ-I cells, but not in NCI-H727 cells. This resulted in a decrease in transcripts for Ki-67 and an elevation of the cell cycle inhibitor, p21WAF1/CIP1. Preincubation with the MEK inhibitor, PD98059, decreased these lanreotide-mediated alterations in p21WAF1/CIP1 expression. In studies in Chinese hamster epithelial ovary CHO-K1cells, lanreotide-stimulated ERK activity led to accumulation of phosphorylated P27Kip1, inhibition of cyclinE-cdk2 kinase activity, accumulation of hypophosphorylated p105Rb, and cell cycle arrest.29, 30 Over-expression of P21WAF1/CIP1, a cyclin-dependent kinase (cdk) inhibitor, leads to G1 and G2 or S-phase arrest.31 Our findings indicate that the inhibitory effects of lanreotide on H720 and KRJ-I are associated with cell cycle arrest, at least via p21WAF1/CIP1 transcriptional up-regulation.

In NCI-H720, BIM23A761 did not phosphorylate ERK1/2, AKT, or NFκB but activated JNK phosphorylation. JNK was also phosphorylated in BIM23A761-treated NCI-H727 cells, but phosphorylation of ERK1/2 was inhibited. Although BIM23A761 caused a decrease in transcripts for Ki-67 in both cell lines, an elevation of the cell cycle inhibitor p21WAF1/CIP1 was only detected in NCI-H727 cells. In contrast, expression of this cdk inhibitor was significantly decreased in NCI-H720 cells. This seemingly discrepant observation raises the issue of the precise role of JNK signaling in apoptosis, because JNK has been reported to function as both a pro- and antiapoptotic agent.32 JNK induces phosphorylation of c-Jun at Ser63, a site important for c-Jun–dependent transcriptional activity,33 and the combination of c-Jun activation and p21WAF1/CIP1 suppression stimulates P53-dependant apoptosis.31 Our finding of suppressed levels of P21WAF1/CIP1 in NCI-H720 cells is consistent with a role for BIM23A761 in inhibiting cell proliferation via a P53-dependent apoptosis. In contrast, BIM23A761-mediated growth inhibition in NCI-H727 cells, similar to lanreotide in NCI-H720 and KRJ-I cells, is associated with P21WAF1/CIP1-mediated cell cycle inhibition through the retinoblastoma pathway.29, 30 It is thus likely that individual NETs will not share completely similar transduction mechanisms for proliferative regulation, and that specific assessments will be necessary to identify and predict the most efficacious therapeutic agent.

Synthetic derivatives of somatostatin, such as octreotide, are in common clinical use as therapy for acromegaly and gastroenteropancreatic tumors.34 Although effective in the inhibition of the secretion of bioactive products and the amelioration of symptomatology,35 they have been disappointing as antiproliferative agents in vivo.7 The reasons for this are not known, but it indicates that secretory regulation of NET cells is an intrinsic property, whereas the proliferative drive may comprise many agents (angiogenic factors, growth factors) and has a substantial extrinsic basis, likely from the tumor micro-milieu.

We have used an in vitro model to study the effects of selective somatostatin and dopamine compounds on NET cells. With the caveat that our studies cannot measure the effects of these compounds on cells, eg, fibroblasts and endothelial cells in the NET microenvironment that regulate NET proliferation and secretion,5, 36, 37 we demonstrate that targeting specific receptor subtypes on tumor cells, with selective somatostatin agonists, may be more advantageous in “atypical” bronchopulmonary NETs than a single generic compound. In contrast, bronchopulmonary NETs and gastrointestinal NETs with an extensive somatostatin receptor expression profile may be more responsive to a pan-somatostatin analog.25 Because some NETs express somatostatin and D2 receptors, it is has been suggested that sst/da chimeric compounds may prove to be valuable in the management of NETs that exhibit both receptor profiles.38 Our results support this observation, because somatostatin/dopamine chimeric compounds are as effective as pan-somatostatin analogs in inhibiting NET cell proliferation. They do, however, appear to be less effective than single receptor targeted somatostatin agonist therapy for “atypical” bronchopulmonary NETs.

In conclusion, individual (site and cell specific) NETs respond differently to specific somatostatin analogs and sst/da chimeric compounds, depending on the somatostatin and dopamine receptor profile coupling to signaling pathways. Antiproliferative effects are because of ERK1/2 phosphorylation and JNK signaling and activation of cdk inhibitors with a decrease in Ki-67 (Fig. 6). If a therapeutic strategy that embraces receptor targeting is used for NETs, it will require prior delineation of the specific receptor profile of the tumor and the selection of “receptor-tailored” pharmacotherapy to optimize effective treatment.

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Figure 6. Somatostatin and sst/da chimera receptor signaling pathways in NET cell lines are shown. Somatostatin receptor activation (largely via sst2) results in MEK activation and downstream phosphorylation of ERK (A). Activated ERK phosphorylates and activates the cdk inhibitor P27KIP1 and up-regulates P21WAF1/CIP1, while inhibiting transcription of the cyclin, Ki-67. The combination of cdk inhibitor up-regulation and a decrease in Ki-67 results in growth arrest. sst/da receptor activation (largely sst2 of 5/D2) results in phosphorylation of JNK (B). Activated JNK up-regulates P21WAF1/CIP1, while inhibiting transcription of the cyclin, Ki-67. This combination results in growth arrest. Depending on the sst/da receptor complex, p21WAF1/CIP1 transcription can also be decreased.

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REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES