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Inhibition of proliferation of small intestinal and bronchopulmonary neuroendocrine cell lines by using peptide analogs targeting receptors
Article first published online: 25 JAN 2008
Copyright © 2008 American Cancer Society
Volume 112, Issue 6, pages 1404–1414, 15 March 2008
How to Cite
Kidd, M., Schally, A. V., Pfragner, R., Malfertheiner, M. V. and Modlin, I. M. (2008), Inhibition of proliferation of small intestinal and bronchopulmonary neuroendocrine cell lines by using peptide analogs targeting receptors. Cancer, 112: 1404–1414. doi: 10.1002/cncr.23303
- Issue published online: 3 MAR 2008
- Article first published online: 25 JAN 2008
- Manuscript Accepted: 12 DEC 2007
- Manuscript Revised: 1 OCT 2007
- Manuscript Received: 2 AUG 2007
- NIH. Grant Number: R01-CA1185285
- Bruggeman Medical Foundation
- bronchopulmonary carcinoid;
- cytotoxic peptide analogs;
- drug sensitivity;
- neuroendocrine tumor;
Currently, no consistently effective therapy is available to inhibit cell proliferation or metastasis of neuroendocrine tumor (NET) disease. The effects of 4 novel peptides were analyzed: a targeted cytotoxic analog of luteinizing hormone-releasing hormone (LH-RH) analog (AN-152), a targeted cytotoxic analog of somatostatin (AN-238), and 2 antagonists of growth hormone-releasing hormone (GH-RH) on 3 NET (carcinoid) cell lines that expressed respective peptide receptors.
The effects of the compounds were evaluated on cell proliferation in vitro using MTT uptake and Ki67 expression, apoptosis (caspase 3 expression and activity), and cell cycle parameters (DNA distribution).
Proliferation of the LH-RH receptor-expressing lung NET, NCI-H720 line, was inhibited 2-fold by AN-152 containing doxorubicin compared with the chemotherapy alone (IC50 of 9.1 nM vs 24 nM). This was associated with a reduction in Ki67 transcript and an increase in both caspase 3 mRNA levels and activity. Proliferation of the GH-RH receptor expressing lung NET, NCI-H727 line, was inhibited by both GH-RH antagonists, the effects being mediated through changes in Ki67 expression, but not in caspase 3-mediated apoptosis. The small intestinal NET, KRJ-I line, was 8× more sensitive to inhibition by AN-238 than to 2-pyrolino-doxorubicin, reflected by increased caspase 3 transcript as well as activity. AN-238-mediated growth inhibition culminated in complete G1 arrest.
The data demonstrate GH-RH antagonists or peptide-linked antineoplastic agents such as AN-152 and AN-238 are effective inhibitors of NET proliferation in vitro. Because peptide receptors such as those for GH-RH, LH-RH, and SST subtypes are commonly expressed by NETs, the development of antineoplastic agents targeted to specific tumor receptors may provide a more efficacious strategy than systemic chemotherapeutic agents currently in use. Cancer 2008. © 2008 American Cancer Society.
Neuroendocrine tumors (NETs) are a group of neoplasms that originate in neuroendocrine cells and are part of the diffuse neuroendocrine system that is spread throughout the gastrointestinal tract and the bronchopulmonary system.1 Overall, NETs are diverse in their specific cells of origin (enterochromaffin, EC-like, etc), secrete a variety of amines and peptides, and present a wide variety of symptoms including flushing, sweating, and diarrhea.2 The SEER database demonstrates that NETs are increasing in incidence at a rate of ≈10% per year and therefore represent a substantial clinical problem.2, 3 The majority of NETs arise in the gastrointestinal tract (67.5%), but a considerable number is present in the bronchopulmonary system (25.3%).3
Although in the past these lesions were considered to exhibit a benign behavior, it is now apparent that this is incorrect and, indeed, the vast majority are metastatic at diagnosis.4 Initial therapy is therefore directed toward surgical removal of the primary and ablation or resection of as much metastatic disease as is safe and feasible.5 Given the relatively indolent biological behavior, conventional radiotherapy is of little value. Similarly, there is no specific chemotherapy and current clinical data indicate that objective tumor responses occur only in ≈10% of patients.5 Novel cytotoxic agents including antiangiogenic drugs, growth factor inhibitors, and tyrosine kinase inhibitors have been studied,6, 7 but their utility is limited and many patients exhibit substantial adverse effects.5 Because the majority of these tumors express somatostatin receptor subtype 2 (SSTR2), to date the best available approach has been the use of long-acting somatostatin analogs such as Somatuline, which are effective in the amelioration of symptomatology.8 Whereas this class of pharmacotherapeutic agents inhibits the secretion of bioactive agents and hence symptoms, it is ineffective in the inhibition of neuroendocrine cell proliferation.2, 5
An approach based on targeted antineoplastic therapy focusing on specific molecular pathways or receptors expressed by malignant cells, while leaving normal cells unaffected, has promise.9, 10 Thus, the observation of increased expression of SSTR2 by NET cells has been proposed for a strategy to use cytotoxic peptide conjugates to selectively deliver specific agents to NETS and their metastases. The linkage of high-energy radioisotopes (111In, 90Y, 177Lu) to somatostatin analogs administered intravenously has been used with some success to treat GEP-NETs.11 Although the ideal isotope remains to be identified and issues with renal and bone marrow toxicity require careful observation, it seems likely that this concept will prove of therapeutic benefit.12
An alternative strategy to peptide receptor radiotherapy (PPRT) is the use of receptor-targeted chemotherapy using specific cytotoxins. The approach of using peptides (eg, luteinizing hormone releasing hormone, LH-RH), somatostatin or bombesin/GRP that target the NET cell and are conjugated to chemotherapeutic agents (eg, doxorubicin) known to have efficacy in NET management13 has been extensively investigated. Experimental studies in human cancer cell lines xenografted into nude mice have indicated that this strategy is feasible and effective.12, 14, 15 Thus, the cytotoxic LH-RH analog, doxorubicin conjugated to AN-152, has been investigated in a variety of cancers (breast, ovarian, endometrial, and prostate cancers) with promising results16 and is now in phase 2 clinical trials in Germany. Preclinical evaluation of the cytotoxic somatostatin analog, AN-238, has demonstrated it to be effective on a wide variety of non-neuroendocrine tumors (melanoma, hepatoma, glioblastomas, non-Hodgkin lymphoma [NHL]).14 Another approach consists of antagonists of growth hormone-releasing hormone (GH-RH), which act on specific tumor receptors for GH-RH and inhibit growth of a broad variety of human experimental tumor models (small-cell lung carcinoma, prostate carcinoma, NHL).17–19 GH-RH antagonists are thought to function through inhibition of autocrine/paracrine production of IGF-I and IGF-II in tumors and/or the blockade of the stimulatory effect of GH-RH in tumors.19 Little is known about the effects of such agents on GEP-NETs.
In this study we investigated whether these peptide analogs might be effective in human neuroendocrine cells and examined the mechanisms by which these classes of agents affect NET cells. We evaluated the cytotoxic LH-RH analog AN-152 and the somatostatin analog AN-238 as well as 2 GH-RH antagonists to assess the inhibition of cell proliferation in 3 NET (carcinoid) cell lines. Our goal was to determine whether GH-RH antagonists could be used for inhibition of NET cell lines and, if so, to assess whether targeting specific LH-RH receptors, SSTR2, 3, and 5 receptors, as well as whether GH-RH antagonists could be used for inhibition of NET cell lines.
MATERIALS AND METHODS
The studies were undertaken in 3 NET cell lines; an atypical lung carcinoid (NCI-H720), a typical bronchial carcinoid (NCI-H727), and a small intestinal carcinoid (KRJ-I).20, 21 The receptor profile of each cell line was first examined using real-time polymerase chain reaction (PCR). MTT uptake (after 72 hours incubation with each agent: 1–100 nM) was used to measure the efficacy of each cytotoxic-analog or GH-RH antagonist to inhibit cell proliferation compared with the cytotoxic radical alone. Inhibition of cell proliferation by IC50 concentrations of each effective agent was confirmed using real-time quantitative PCR and the normalization of expression levels of the target gene, Ki67, using geNorm and 3 housekeeping genes (ALG9, TFCP2, and ZNF410).22 Apoptotic pathway activation after treatment with the IC50 concentrations of each agent was evaluated by PCR analysis of caspase 3 transcript and measurement of caspase 3 activity using the PhiPhiLux assay.23, 24 Finally, flow cytometric analysis was performed and cell cycle alterations were examined in response to IC50 concentrations of each compound over a 72-hour period (2 population doubling times for each cell line20, 21). The requirement for trypsin digest with the associated cell death in the adherent NCI-H727 cell line precluded flow cytometric analysis.
Cytotoxic LH-RH conjugate AN-152 consists of LH-RH linked to doxorubicin (Dox).25 Dox hydrochloride was obtained from Chemex Export-Import (Vienna, Austria). The SST analog RC-121 was coupled with 1 molecule of 2-pyrrolinodoxorubicin-14-0-hemiglutarate (AN-201) to form AN-238.26 AN-201 (Dox2) was synthesized by a conversion of Dox to 3′-deamino-3′-(2″-pyrroline-1″-yl) doxorubicin.25
KRJ-I and NCI-H720 cells were cultured as floating aggregates at 37°C with 5% CO2. KRJ-I cells were kept in Ham F12 medium (Gibco, Gaithersburg, Md) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, Mo), penicillin 100 U/mL, and streptomycin (100 μg/mL).21, 29, 30 For NCI-H720, a 1:1 solution of Ham F12 and Dulbecco minimal essential medium (DMEM) 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), HEPES medium (10 mM), and L-glutamine (2 mM) was used.20 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).20 For subculturing, NCI-H727 cells were washed in phosphate-buffered saline (PBS) before trypsin-EDTA was applied.
After centrifugation (5 minutes; 1500g) pellets were resuspended in respective media to a concentration of 5 × 105 cells/mL and seeded in 96-well plates at 100 μL with 5 × 104 cells/well (4 plates/experimental condition).29, 30 Lanes 1 and 2 contained negative (medium only) and positive (cell suspension) controls. Drugs were diluted in PBS and applied at final concentrations from 1 nM to 100 nM (n = 6 wells for each concentration/plate).
After 72 hours of incubation (37°C; 5% CO2), 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.29-31 The reaction was stopped and the formazan dye solubilized by adding an equal volume (100 μL) of acid-isopropanol (0.01 N HCl-isopropanol). The optical density was read at 595 nm using a microplate reader (Bio-Rad 3500, Hercules, Calif).29, 30 Values were averaged in each of the 6 treated wells and values for media alone (lane 1) were subtracted. These values were then normalized to control (unstimulated cells, lane 2) and used to calculate the effective half-maximal concentrations for each drug as well as the percentage of cell death (treated values, normalized to control × 100).29, 30
RNA Extraction and Reverse Transcription
Total RNA was extracted from each cell line (n = 4) using TRIZOL (Invitrogen, La Jolla, Calif) and the RNA quality assessed using Agilent Bioanalyzer (Agilent Technologies, Palo Alto, Calif) to visually verify the absence of genomic DNA contamination, integrity and ratio of 28S and 18S bands. Samples with A260/A280 ratio ≥1.8 were used for PCR.22, 29 Total RNA (2 μg) from each preparation was reverse-transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, Calif).
Quantitative Real-Time Polymerase Chain Reaction (Q RT-PCR)
Q RT-PCR was performed using the ABI 7900 Sequence Detection System. Briefly, cDNA in 7.2 μL of water was mixed with 0.8 μL of 20 × Assays-on-Demand primer (caspase 3, Ki-67, LHRH-R, the pituitary type GHRH-R and SSTR 1-5) and probe mix, 8 μL of 2 × TaqMan Universal Master mix in a 384-well optical reaction plate. The following PCR conditions were used: 50°C for 2 minutes, then 95°C for 10 minutes, followed by 40 cycles at 95°C / 0.15 minutes and 60°C / 1 minute.30 Data were normalized using geNorm32 and expression of the novel housekeeping genes, ALG9, TFCP2, and ZNF410.22, 30
Detection of Caspase-3 Activity by FACS
For the analysis of caspase-3 activity, cells were plated in 12 well plates (1 × 106 cells/well in 1 mL) and treated for 21 hours with IC50 values (from the proliferation studies) of each agent of interest. Caspase-3 activity was evaluated using the PhiPhiLux G1D2 kit (MBL, Nagoya, Japan) as described.23, 24 A substrate of PhiPhiLux G1D2, which can penetrate into the cell nucleus, is converted to the fluorescent form when cleaved by the protease activity of caspase-3. The assessment was performed according to the manufacturer's recommendations. Briefly, cells were incubated with 75 μL of PhiPhiLux G1D2 for 1 hour at 37°C in 5% CO2, and caspase-3 activity analyzed using fluorescence-activated cell sorting caliber flow cytometry as described.23, 24
Cell Cycle Analysis
For the analysis of the cell cycle, cells were plated in 12 well plates (1 × 106 cells/well in 1 mL) and treated for 24, 48, and 72 hours with IC50 values (from the proliferation studies) of each agent of interest. At the end of the experiment cells were fixed in 1 mL 75% ethanol and maintained at room temperature. For cell cycle analysis, fixed cells were resuspended in PBS and stained with propidium iodide (PI, 5 mg/mL) immediately before FACS analysis (BD FACS Aria, Yale Cancer Center).33 Cell cycle alterations were analyzed using FloJo Software (TreeStar, Calif) and the Dean-Jett-Fox algorithm.33
All statistical analyses were performed using Microsoft Excel and Prism 4 (GraphPad Software, San Diego, Calif).
Real-Time PCR Receptor Profile
Analysis of receptor levels by real-time PCR (Fig. 1) in each of the cell lines demonstrated that the LH-RH receptor (LH-RH-R) was detectable in the atypical lung carcinoid cell line NCI-H720 (Fig. 1B), whereas the pituitary type GH-RH receptor (GH-RH-R) was expressed only in the bronchial carcinoid cell line NCI-H727 (Fig. 1A). Neither receptor was identified in the small intestinal KRJ-I cell line. Somatostatin receptors (SSTR) also demonstrated a variable expression profile. NCI-H720 exhibited low expression of SSTR2 and R5 and no expression of SSTR1 and R3, whereas SSTR5 had the highest expression level in NCI-H727, followed by SSTR1, R3, and R2. KRJ-I was positive for SSTR1-3 and R5. SSTR4 was never detected (data not shown).
Antiproliferative Effects of AN-152, AN-238, and the GH-RH Antagonists
The LH-RH-doxorubicin analog (IC50 9.1 nM) was >2× more effective than Dox alone (Fig. 2A) at inhibiting MTT uptake in the NCI-H720 cell line. The SST analog linked to Dox2 was not effective for inhibiting cell proliferation (Fig. 2B). MZ-5-156 was a potent inhibitor of NCI-H720 proliferation but MZ-4-71 demonstrated no response (Fig. 2C).
No differences were noted between the LH-RH-doxorubicin analog and Dox alone (Fig. 2D), whereas the dox2 and SST analog (AN-238) were likewise ineffective (Fig. 2E). However, targeting the GH-RH-R with MZ-5-156 induced a potent inhibition of NCI-H727 proliferation (Fig. 2F). As for NCI-H720, MZ-4-71 had no effect on this cell line.
Effects of Inhibitors on Transcript Levels of Ki67 and Caspase 3
Both AN-152 and Dox significantly (P < .05) inhibited transcript levels of Ki67 in NCI-H720 cells compared with untreated cells (control), demonstrating that decreased Ki67 transcription characterized inhibition of cell proliferation (Fig. 3A). In contrast, caspase 3 transcripts were significantly elevated by both AN-152 and Dox in NCI-H720 cells (Fig. 3D), indicating that these agents activate this apoptotic pathway in this cell line.
MZ-5-156 significantly inhibited Ki67 levels compared with both MZ-4-71 (P = .02) and untreated cells (P = .01) (Fig. 3B). In contrast, neither MZ-4-71 nor MZ-5-156 altered caspase 3 transcripts in NCI-H727 cells (Fig. 3E), suggesting that this apoptotic pathway was not activated by these GH-RH receptor antagonists.
Caspase 3-Mediated Activity and Apoptosis
In NCI-H720 cells, AN-152 was significantly more potent in activating caspase 3 compared with either Dox alone (P = .006) or control (P = .0002) (Fig. 4A). In addition, the number of unstained (live) cells was significantly reduced by AN-152 compared with the control cells (Fig. 4B). We found no significant differences in the proportions of unstained cells (live cells) or the double-stained (PI and PhiPhiLux) cells between controls or cells treated with either AN-152 or Dox cells.
Cell Cycle Analyses
In NCI-H720 cells, AN-152 caused a decrease in the growth rate that was evident as early as 48 hours (Fig. 5B).
In KRJ-I cells, AN-238 caused a decrease in the growth rate that culminated in a complete G1 arrest between 48-72 hours (Fig. 5E,F). Dox2 was not associated with any significant alteration in the cell cycle.
Therapy for NETs is currently limited because no specific therapy is available to inhibit neuroendocrine cell proliferation and metastasis. Our investigation of 3 human neuroendocrine cell lines demonstrates that cytotoxic agents linked to analogs that target specific NET receptors provide an effective mechanism for inhibiting NET proliferation and inducing apoptosis in vitro. It was notable that each cell line responded to different agents. This suggests that individual tumor receptor profiling may be required to identify the most appropriate and relevant target in a particular lesion and to adapt the appropriate therapy for each patient with NETs.
LH-RH Analog: AN-152
The atypical lung NET (NCI-H720) that expressed LH-RH-R was more responsive to Dox linked to an LH-RH analog (AN-152) than to Dox alone. The 2 other cell lines (a typical lung and small intestinal carcinoid/NET) showed no significant inhibitory response to this agent. This is consistent with the observation that neither of them expressed a transcript for this receptor. AN-152 was twice as effective as Dox alone (IC50 of 9.1 nM vs 24 nM) and killed more NCI-H720 cells (30% cell death after 72 hours vs 20%). This was confirmed by real-time PCR, which demonstrated a reduction of Ki67 transcript. The increase in caspase 3 mRNA levels was mirrored by an increased caspase 3 activity that demonstrates that a mechanism of AN-152 cytotoxicity is induction of apoptosis (Table 1). These effects were complemented by an overall decrease in the growth rate of these cells (cell cycle analysis) confirming inhibition of proliferation. AN-152 has previously been shown to be an effective inhibitor of tumor growth in breast, prostate, endometrial, and ovarian cancers.16 In addition, because LH-RH-R is known to be expressed at very low levels on most normal tissues apart from the pituitary,34 targeting this receptor, particularly in NETs with high receptor expression, suggests that the LH-RH analog is a potentially useful target for receptor targeted chemotherapy. Clinical phase 1 trials with AN-152 in women with gynecologic tumors have been completed and phase 2 is in progress.
|Cell line||Receptor profile||MTT assay inhibitors||Ki67 mRNA||Caspase 3 mRNA||Caspase 3 activity||% Live cells||Cell cycle|
|NCI-H720 Atypical lung NET||LH-RH R SSTR2/5*||AN152 MZ-5-156|
|NCI-H727 Typical lung NET||GH-RH R SSTR1,2,3,5||MZ-5-156 MZ-4-71||NA||NA||NA|
|KRJ-I Typical GI NET||SSTR1,2,3,5||AN-238||G1 arrest|
SST Analog: AN-238
It has previously been reported that the antitumoral effect of AN-238 is essentially equal in xenografted human tumors that express either SSTR2 or SSTR5 (and produced a similar reduction in tumor volume: 67% vs 78%).35 The SST receptor expression profiles we identified (Fig. 1) suggest that AN-238 would be most effective on KRJ-I and NCI-H727 cells. NCI-H720 expresses lower levels of the type 2 and 5 SST receptors and does not express the other subtypes. AN-238 was identified to be an effective inhibitor of KRJ-I proliferation (8× as effective as doxorubicin alone) and the degree of cell inhibition was substantially higher than for either NCI-H727 or NCI-H720, which were not preferentially inhibited by this agent compared with the chemotherapeutic radical alone. In addition, AN-238 increased both caspase 3 transcripts and caspase 3 activity and caused a decrease in KRJ-I growth that culminated in complete G1 arrest (Table 1). Doxorubicin is an anthracycline that inhibits topoisomerase II by formation of a tripartite complex with the enzyme, which prevents the re- ligation of broken DNA strands and finally leads to apoptosis.36 Doxorubicin is also known to disrupt the S-phase of the cell cycle. Although somatostatin is considered to activate cyclin inhibitors, particularly P27KIP1, which is recognized to inhibit G0/G1 and cause cell cycle arrest,37 the inhibition of KRJ-I proliferation probably reflects an effect only of targeted doxorubicin because the dose of somatostatin analog RC-121 contained in the conjugate was low (0.5 nM).
Clinical studies in patients with GEP-NETs have provided only minimal evidence for an antitumorigenic role for somatostatin analogs.38 Whereas somatostatin effectively regulates carcinoid symptomatology (through inhibition of secretory products),5, 8 a role in inhibiting tumor growth in vivo remains to be proven. Doxorubicin itself is of modest and unpredictable efficacy (16%-20% objective therapeutic response) in the treatment of NETs.39, 40 Our current study indicates that a hybrid of a somatostatin analog RC-121 and doxorubicin targeted to SST receptors may provide an effective mechanism for treating GEP-NET cells.
GH-RH Antagonists: MZ-4-71 and MZ-5-156
GH-RH antagonists were developed for inhibition of tumor growth because somatostatin analogs did not have adequate oncologic activity.17, 18, 41 The findings that extrapituitary tumors (eg, prostatic, mammary, renal, NHL, lung, pancreatic, and ovarian cancers) express GH-RH-R splice variants has extended the types of neoplasia that could be treated by such antagonists.42 We demonstrated that the typical lung NET cell line, NCI-H727, transcribed GH-RH-R, which suggested that the GH-RH antagonists could potentially inhibit proliferation of these cells but would have no effect on NCI-H720 or KRJ-I, which do not express transcripts for the receptor.
Both MZ-4-71 and especially the more potent MZ-5-156 had an inhibitory effect on NCI-H727 but not on KRJ-I. Moreover, whereas MZ-4-71 had no effect on NCI-H720, MZ-5-156 did inhibit cell proliferation (Table 1). This finding in a tumor cell line that did not express transcripts for the pituitary type of GH-RH receptor could be due either to the inability of real-time PCR to identify all splice variants of the receptor or to potential crossreactivity with another receptor. Because of the difference in primers, the expression of SV-type receptors in tumors that bind GH-RH antagonists is more common than that of pGH-RH receptors. Another splice variant of the GH-RH receptor that cannot signal through the cAMP pathway and acts as a dominant inhibitor of wildtype receptor signaling has also been identified.43 The probes we used to identify GH-RH receptor expression do not detect these particular variants. Given the difficulties with adherent cell subculturing and the potential apoptotic effects from trypsin digestion protocols, we were not able to evaluate whether the effects of GH-RH-R antagonists were due to apoptosis or to alterations in the cell cycle in H727 cells. Our evaluation of transcripts for Ki67 and caspase 3, however, suggested that an alteration in the cell cycle, rather than caspase 3-mediated apoptosis, was probably the inhibitory mechanism of these agents in this cell line.
In summary, our data demonstrate that chemotherapeutics linked to peptides have the capability to be effective inhibitors of NET cell proliferation. Thus, a NET cell expressing the appropriate receptor for the peptide carrier provides a target that permits the binding of the hybrid-containing chemotherapeutic agent to the tumor and makes it more effective than when delivered alone. It is thus feasible that, given the presence of various peptide receptors like GH-RH-R, LH-RH-R, and SSTR subtypes in NETs, receptor targeted chemotherapy using a peptide vector based may offer a clinical advantage. Such targeted therapy to neuroendocrine cells would be predicted to have more efficacy and less toxicity than the systemic chemotherapeutic agents currently in use.5 Because appropriate receptors of individual tumors can be selectively identified, an escalation of dose may be achievable with predictably better outcome. To optimize such therapy, the cell biology of individual tumors would have to be characterized based on biopsy material, and once a profile was established in a patient a tumor-specific cytotoxic hybrid or GH-RH antagonist could be administered.
- 17Suppression of growth of H-69 small cell lung carcinoma by antagonists of growth hormone releasing hormone and bombesin is associated with an inhibition of protein kinase C signaling. Int J Cancer. 2004; 112: 570–576., , , , .
- 30Small bowel carcinoid (enterochromaffin cell) neoplasia exhibits transforming growth factor-beta1-mediated regulatory abnormalities including up-regulation of C-Myc and MTA1. Cancer. 2007; 109: 2420–2431., , , et al.
- 33DNA content determination. In: DiamondR,DeMaggioS, eds. In Living Color. Protocols in Flow Cytometry and Cell Sorting: Springer Lab Manual. New York: Springer; 2000: 361..
- 36Goodman & Gilman's The Pharmacological Basis of Therapeutics. 11th ed. New York: McGraw-Hill., , .