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Targeted therapy with a cytotoxic somatostatin analog, AN-238, inhibits growth of human experimental endometrial carcinomas expressing multidrug resistance protein MDR-1†
Version of Record online: 26 JUL 2005
Copyright © 2005 American Cancer Society
Volume 104, Issue 6, pages 1312–1321, 15 September 2005
How to Cite
Engel, J. B., Schally, A. V., Halmos, G., Baker, B., Nagy, A. and Keller, G. (2005), Targeted therapy with a cytotoxic somatostatin analog, AN-238, inhibits growth of human experimental endometrial carcinomas expressing multidrug resistance protein MDR-1. Cancer, 104: 1312–1321. doi: 10.1002/cncr.21327
The current study is dedicated to the late Ana-Maria Comaru-Schally, M.D., who died recently of thyroid carcinoma, for her intellectual, spiritual and personal contribution and for the inspiration she provided to this project.
- Issue online: 31 AUG 2005
- Version of Record online: 26 JUL 2005
- Manuscript Accepted: 4 APR 2005
- Manuscript Revised: 23 MAR 2005
- Manuscript Received: 22 FEB 2005
- Medical Research Service of the Veterans Affairs Department
- ZENTARIS Gmbh (Frankfurt am Main, Germany)
- German Academic Exchange Service (DAAD)
- targeted chemotherapy;
- endometrial carcinoma;
- somatostatin receptor;
- cytotoxic conjugate AN-238;
- multidrug resistance
Chemoresistance mediated by membrane transporters such as multidrug resistance (MDR-1) glycoprotein remains a challenge in the chemotherapy treatment of advanced or recurrent endometrial carcinoma. Targeted chemotherapy might overcome this resistance. The cytotoxic somatostatin (SST) analog, AN-238, consists of a superactive derivative of doxorubicin (DOX), 2-pyrrolino-DOX (AN-201), linked to the SST analog carrier, RC-121. This conjugate binds strongly to SST receptor subtypes (sst) 2a (sst2a) and 5 (sst5) and can be targeted to tumors that express these receptors.
The presence of sst2a and sst5 was determined in 3 human endometrial carcinoma cell lines (HEC-1A, RL-95-2, and AN3CA). Nude mice bearing xenografts of these cancers were treated with AN-238 and its radical, AN-201. The antitumor effects and toxicity were compared. The authors studied the effects of AN-238 and AN-201 on the expression levels of MDR-1, multidrug resistance related protein (MRP-1), and breast carcinoma resistance protein (BCRP) by real-time polymerase chain reaction.
The authors demonstrated the presence of mRNA and receptor protein for sst2a and sst5 on HEC-1A, RL-95-2, and AN3CA tumors. AN-238 significantly (P < 0.05) inhibited the growth of these tumors, whereas AN-201 had no effect. Blockade of SST receptors nullified the effects of AN-238. In all 3 endometrial carcinoma lines, AN-238 caused a weaker induction of MDR-1 than AN-201. No major induction of MRP-1 and BCRP occurred after treatment with AN-238 or AN-201.
Targeted chemotherapy with the cytotoxic SST analog, AN-238, inhibited powerfully the growth of endometrial carcinoma, which express SST receptors, regardless of their expression level of MDR-1. Cancer 2005. © 2005 American Cancer Society.
Endometrial carcinoma is the most common neoplasm of the female genital tract and it accounts for nearly one-half of all gynecologic carcinomas in the United States. It is estimated that approximately 41,000 new cases of endometrial carcinoma will be diagnosed in 2005 and 7300 patients will die of this disease. Thus, in the United States, endometrial carcinoma ranks as the fourth most common neoplasm and the eighth leading cause of death from cancer in females.1–5
If the endometrial tumor is confined to the uterus, the primary treatment is usually surgery followed by radiotherapy.2–5 For patients with advanced-stage or recurrent disease, who have already received tumor debulking and/or radiotherapy, hormonal treatment with progestins or combination chemotherapy is administered.2–5
If discovered at an early stage, patients with endometrial carcinoma have a good prognosis and the 5-year survival rates are 87% and 76% for International Federation of Gynecology and Obstetrics (FIGO) Stages I and II, respectively.5 However, for patients with FIGO Stages III and IV disease, the 5-year survival rates decrease substantially to 59% and 18%, respectively.5 Patients with recurrent endometrial carcinoma face a dismal prognosis with an overall survival of only 7.7%.6 Consequently, new therapeutic strategies are required for the treatment of late-stage and recurrent endometrial carcinoma.
The discovery of specific molecular characteristics of malignant cells prompted the development of a new class of drugs that can be targeted to surface structures on tumor cells. These modern antitumor agents known as targeted therapeutics include monoclonal antibodies and conjugates consisting of receptor-specific ligands linked to toxins, radionuclides, or chemotherapeutic agents.7–10 Malignant cells, which express receptors for peptide hormones, can be targeted using cytotoxic hormone analogs consisting of a peptide carrier linked to a chemotherapeutic radical.11–14 A direct delivery of the antineoplastic agent to tumor cells by these targeted peptide conjugates should result in a higher antitumor activity with a reduced systemic toxicity. In recent years, we developed a series of targeted cytotoxic conjugates based on analogs of somatostatin (SST), luteinizing hormone-releasing hormone (LHRH), or bombesin peptide carriers.11–14
Much evidence indicates that radiolabeled SST analogs are useful for the localization of various malignancies.15 Similarly, analogs of SST can be effective carriers for targeted delivery of cytotoxic radicals and radionuclides for cancer therapy.16–18 Thus, we developed a cytotoxic SST analog, AN-238, which consists of the SST carrier octapeptide RC-121 (D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2) linked covalently to a highly potent derivative of doxorubicin (DOX), 2-pyrrolino-DOX (AN-201). This analog, which fully retains the cytotoxic activity of the radical and the receptor binding affinity of the peptide carrier,19 has been shown to strongly inhibit the growth of various tumors that express SST receptor subtypes 2a, 3, and 5 (sst2a,3,5).20–25
Among human tumors xenografted into nude mice inhibited by therapy with AN-238 were various breast, ovarian, renal, prostatic, lung, brain, pancreatic, colorectal, and gastric carcinoma lines.13, 14, 18 However, no studies have been carried out so far with endometrial tumors. The presence of SST receptors was demonstrated in peritumoral vessels of 4 of 10 human endometrial tumor specimens using 125I-[Tyr3]-octreotide as a radioligand.26 In a recent study, sst2a and sst3 were detected in 43% and 39% of human endometrial tumor specimens, respectively.27 Thus, endometrial tumors might respond to the targeted cytotoxic SST analog, AN-238, which might be more efficacious than systemic chemotherapy.
Multidrug resistance (MDR) in cancer cells is the simultaneous development of resistance to a variety of antitumor agents that appear to be structurally and mechanistically unrelated. One type of MDR is characterized by the enhanced cellular efflux of chemotherapeutic drugs. The product of the MDR-1 gene, an adenosine 5′-triphosphate (ATP)-dependent membrane transporter termed P-glycoprotein (Pgp),28 and the recently discovered MDR related protein-1 (MRP-1), which was also found to be a member of the ATP-binding cassette superfamily of transporter genes, use this mechanism of action.29 Another overlapping, but discrete resistance MDR phenotype, is associated with increased expression of the efflux transporter breast cancer resistance protein (BCRP).30 In previous studies, expression of MDR-1 has been found in most endometrial tumor specimens and in normal endometrial tissue specimens.31–33 MRP immunoreactivity was detected in normal endometrium and it showed a progressive increase from endometrial hyperplasia to endometrial carcinoma, where it was expressed at high levels in > 50% of the patients.34 In that study, a strong MRP expression was linked with a poor survival rate in patients with advanced-stage disease.34 Targeted therapy could be useful for overcoming MDR based on drug efflux pump proteins, by increasing the local concentration of the chemotherapeutic drug and producing a higher intracellular steady-state level of the cytotoxic agent.
In the current study, we evaluated the antitumor activity of the cytotoxic SST analog, AN-238, in vivo, using models of human endometrial carcinoma with different levels of MDR-1 and MRP-1 expression. To investigate the effects of targeted therapy on the development of secondary MDR, we examined the effects of AN-238 and its nontargeted cytotoxic radical on the expression levels of MDR-1, MRP-1, and BCRP.
MATERIALS AND METHODS
Peptides and Cytotoxic Radical
The carrier somatostatin octapeptide, RC-121 (D–Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2), and the cytotoxic radical, AN-201, were synthesized in our laboratory as described.12, 35 AN-238 was made by coupling AN-201-14-O-hemiglutarate to the aminoterminus of [Lys-(N-(9-fluorenyl/methoxycarbonyl)5]RC-121, followed by deprotection and purification.19 For the intravenous injection, the compounds were dissolved in 20 μL of 0.01N acetic acid and diluted with 5% (w/v) aqueous D-mannitol solution (Sigma, Aldrich, St. Louis, MO).
The human endometrial cancer cell line, HEC-1A, is a moderately well differentiated (Grade II) endometrial adenocarcinoma originating from a 71-year-old woman.36 The human endometrial carcinoma cell line, RL-95-2, is an estrogen receptor (ER)-positive adenocarcinoma derived from the primary tumor of a 65-year-old Caucasian woman.37 The undifferentiated, very aggressive AN3CA cell line was derived from a lymph node of a 55-year-old Caucasian woman with primary endometrial carcinoma.38 All cell lines were obtained from American Type Culture Collection (Manassas, VA).
The cells were grown at 37 °C in a humidified 95% air 5% carbon dioxide atmosphere, passaged weekly, and routinely monitored for mycoplasma contamination using a detection kit (Boehringer Mannheim, Mannheim, Germany). All culture media were purchased from Gibco (Grand Island, NY).
Five to 6-week-old female athymic nude mice (Ncr nu/nu) were obtained from the National Cancer Institute (Bethesda, MD). The animals were housed in sterile cages under laminar flow hoods in a temperature-controlled room with a 12-hour light/12-hour dark schedule. They were fed autoclaved chow and water ad libitum.
The cells of each cell line growing exponentially were implanted into 5 donor female nude mice by subcutaneous injection of 107 cells in both flanks. Tumors resulting after 4 weeks in donor animals were aseptically dissected and mechanically minced. In all experiments, 3-mm3 pieces of tumor tissue specimens were transplanted subcutaneously into the experimental animals by a trocar needle. In all the experiments, animals in the experimental groups received a single intravenous injection of the compounds. Tumor volume (length × width × height × 0.5236) and body weight were measured weekly. The total leukocyte count was determined with the Unopette microcollection kit (Becton Dickinson, Franklin Lakes, NJ).
At the end of each experiment, mice were killed under anesthesia, tumors were excised and weighed, and necropsy was performed. Tumor specimens were snap frozen and stored at −70 °C.
All experiments were performed in accordance with institutional guidelines for the welfare of animals in experiments. The Institutional Animal Care and Use Committee reviewed the protocols of the animal experiments and gave full approval.
In Experiment 1, when HEC-1A tumors had reached a volume of approximately 60 mm3, mice were assigned to five experimental groups: Group 1, control mice received vehicle solution (10 mice); Group 2, mice were given the cytotoxic analog, AN-238, at 200 nmol/kg (10 mice); Group 3, mice were injected with the cytotoxic radical, AN-201, at 200 nmol/kg (10 mice); Group 4, mice received a mixture of the cytotoxic radical, AN-201, and the carrier RC-121 at 200 nmol/kg (5 mice); and Group 5, mice received RC-121 at 200 nmol/kg (5 mice).
In Experiment 2, when RL-95-2 tumors had reached a volume of approximately 70–75 mm3, mice were divided into 4 experimental groups: Group 1, control mice were injected with vehicle solution (10 mice); Group 2, mice received the cytotoxic analog, AN-238, at 200 nmol/kg (10 mice); Group 3, mice were given the cytotoxic radical, AN-201, at 200 nmol/kg (10 mice); Group 4, mice received 200 μg of the SST analog, RC-160, intravenously 15 minutes before the the intravenous injection of the cytotoxic analog, AN-238, at 200 nmol/kg (5 mice).
In Experiment 3, when AN3CA tumors had reached a volume of approximately 140 mm3, mice were assigned to 3 experimental groups: Group 1, control mice were given vehicle solution (11 mice); Group 2, mice were injected with the cytotoxic analog, AN-238, at 200 nmol/kg (11 mice); Group 3, mice received the cytotoxic radical, AN-201, at 200 nmol/kg (10 mice).
Healthy nude mice were injected with control vehicle solution, AN-238, or AN-201 at 200 nmol/kg. On Day 5, mice were killed and their kidneys were excised and fixed in 10% buffered formalin for histologic studies.
Fixed specimens were embedded in paraplast (Oxford Labware, St. Louis, MO), and 6-μm thick sections of the kidney specimens were cut and stained with hematoxylin and eosin. Specimens were examined by light microscopy for any kind of cellular damage.
RNA Extraction and Reverse Transcription-Polymerase Chain Reaction
RNA was isolated from approximately 100 mg of tumor tissue specimen for each sample according to the TRI-Reagent protocol (Sigma-Aldrich), following the manufacturer's instructions. The reverse transcription (RT) reaction was performed with the iScript cDNA synthesis kit from Bio-Rad (Hercules, CA). The RT reaction was performed according to the manufacturer's instructions. One microgram of RNA was transcribed into cDNA in a final volume of 10 μL. All polymerase chain reactions (PCR) were performed in an Applied Biosystems PCR system 2700 (Applied Biosystems, Norwalk, CT). For the amplification of the cDNA, gene-specific primers for sst1, sst2a, sst3, sst4, and sst5 were used as described.39 A total of 10 μL of each PCR product was loaded on a 1.8% agarose gel and subjected to electrophoresis, staining with ethidium bromide, and analysis using Kodak 1D 3.6 imaging analysis software (Kodak, Rochester, NY). A total RNA negative control, which had only water in the RT reaction, was used in each PCR to rule out genomic DNA contamination. The PC-3 human prostate carcinoma cell line was used as a positive control in the PCR.
Western Immunoblot Analysis
For immunodetection of sst2 and sst5, an extraction of membrane protein from untreated HEC-1A, RL-95-2, and AN3CA samples was performed as reported.40–42 The presence of sst2 and sst5 receptor protein was then demonstrated by Western blotting using 2 goat polyclonal human sst2 and sst5 antibodies at a dilution of 1:250 (Santa Cruz Biotechnology, Santa Cruz, CA), as described.42
Receptor Binding Studies
Binding sites for SST on HEC-1A, RL-95-2, and AN3CA tumor specimens from the control groups were characterized by the ligand competition assay. Preparation of tumor membrane fractions and receptor binding studies of SST were performed as described.39, 43 The LIGAND-PC computerized curve-fitting program of Munson and Rodbard44 was used to determine the type of receptor binding, dissociation constant (Kd), and maximal binding capacity of the receptors (Bmax).
Real-Time Polymerase Chain Reaction for MDR-1, MRP-1, and BCRP mRNA Expression
Total RNA was isolated from approximately 100 mg of tumor tissue specimen for each sample according to the TRI-Reagent protocol. One microgram of total RNA was subjected to RT with the Iscript cDNA synthesis kit (Bio-Rad) following the manufacturer's protocol. Real-time PCR was employed to measure drug resistance gene expression using the SYBR Green system (Bio-Rad). Primers for MDR-1 [sense 5′-TCT GGA GGA AGA CAT GAC CAG GTA-3′; antisense 5′-GGC ACC AAA ATG AAA CCT GAA TGT-3′], MRP-1 [sense 5′-AGA GAC AGC TCA GCA GCT CCT-3′; antisense 5′-GCC TTG TCA GCC TCC ATC AG-3′], BCRP [sense 5′-TAT CAA TGG GAT CAT GAA ACC TGG-3′, antisense: 5′-GCG GTG CTC CAT TTA TCA GAA C-3′], and β-actin [sense 5′-CTG GAA CGG TGA AGG TGA CA-3′; antisense 5′-AAG GGA CTT CCT GTA ACA ATG CA-3′] were used to measure gene expression. The thermal cycling conditions comprised an initial denaturation step at 95 °C for 3 minutes, then 40 cycles of 2-step PCR including 95 °C for 15 seconds and 60 °C for 1 minute. Data were collected during the 60 °C annealing step and were further analyzed by the i-Cycler iQ Optical system software (Bio-Rad). Real-time PCR efficiencies (E) for MDR-1 (target gene 1), MRP (target gene 2) and β-actin (reference gene) were calculated from the given slopes in the i-Cycler software according to the following equation : E = 10 [−1/slope].45 Nine tumor samples from each experiment (3 control, 3 AN-238, and 3 AN-201) were analyzed in triplicate. For the mathematical model used in the current study, it was necessary to determine the crossing points (CPs) for the transcripts of each sample. CPs are defined as the number of cycles at which the fluorescense increases appreciably above the background fluorescence. Using CP deviations (ΔCP) for control and treatment (CPcontrols − CPtreatment) of target and reference gene transcripts, quantification of the target genes in treated groups relative to the controls was performed using a mathematical model by Paffl46: ratio = (Etarget)Δ CP target (control − treatment): (Ereference)Δ CP reference (control − treatment).
Data are expressed as the means ± standard error. Differences between mean values were evaluated by the two-tailed Student's t test. P < 0.05 was considered significant.
Effects of Cytotoxic Compounds on Tumor Growth In Vivo
In Experiment 1, a single injection of 200 nmol/kg of the cytotoxic SST analog, AN-238, significantly inhibited the growth of ER-negative HEC-1A human endometrial tumors from Day 8 until the end of the experiment on Day 29. In animals treated with AN-238, tumor volume and weight were significantly reduced by 52.8% and 56.2% (P < 0.05), respectively. The tumor doubling time was also significantly prolonged (P < 0.05) by AN-238 to 40.6 days from 16.9 days in controls. An equimolar dose of the cytotoxic radical, AN-201, a mixture of the carrier RC-121 and AN-201, and RC-121 alone had no significant effects on any growth characteristics (Fig. 1 and Table 1).
|Experiment no. and cell line||Treatment||Initial tumor volume in mm3||Final tumor volume in mm3 (% inhibition)||Tumor weight in mg (% inhibition)||Tumor doubling time in days||Leukocyte count in cells/mm3 on Day 8||Death in experimental groups|
|1. HEC-1A||control||65.1 ± 10.3||248.7 ± 52.4||290.8 ± 58.2||16.9 ± 1.7||8443 ± 939||—|
|AN-238||61.5 ± 9.4||117.3 ± 33.8 (52.8)b||127.3 ± 26.9 (56.2)b||40.6 ± 7.7c||5913 ± 281.8||—|
|AN-201||63.5 ± 9.8||209.3 ± 63.4 (15.8)||225.4 ± 65.5 (22.5)||22.2 ± 2.5||4428 ± 358b||—|
|Unconjugated mixture||65.6 ± 17.7||176.2 ± 63.4 (29.2)||193.9 ± 68.0 (33.3)||29.7 ± 7.5||ND||—|
|Carrier||62.3 ± 13.3||238.8 ± 60.8 (4.0)||253.3 ± 101.8 (12.9)||20.8 ± 2.7||ND||—|
|2. RL-95-2||control||75.9 ± 16.6||455.8 ± 121.5||513.7 ± 129.5||12.6 ± 0.9||9955 ± 819||1/10|
|AN-238||72.0 ± 14.2||166.2 ± 31.3 (63.5)b||206.5 ± 39.3 (59.8)b||24.4 ± 3.6c||7040 ± 1049||1/10|
|AN-201||77.6 ± 18.6||337.1 ± 111.2 (26.0)||416.1 ± 142.1 (19.0)||15.1 ± 1.1||5445 ± 670b||4/10|
|Blockade||74.2 ± 30.7||386.6 ± 189.5 (15.2)||455.3 ± 201.4 (11.4)||17.4 ± 3.3||ND||1/5|
|3. AN3CA||control||137.9 ± 26.8||3812.1 ± 656.5||4768.5 ± 982.2||4.9 ± 0.4||10918 ± 567||1/11|
|AN-238||137.5 ± 22.1||1644.3 ± 222.9 (56.9)b||1929.5 ± 240.3 (59.5)b||6.4 ± 0.5b||7618 ± 1525||—|
|AN-201||133.1 ± 28.8||3889.0 ± 879.9 (+2.0)||4981.5 ± 1090.2 (+4.5)||4.9 ± 0.4||5720 ± 273c||—|
In Experiment 2, a single dose of 200 nmol/kg of the cytotoxic SST analog, AN-238, significantly inhibited the growth of ER-negative RL-95-2 human endometrial tumors as reflected by a tumor volume and weight decrease of 63.5 % (P < 0.05) and 59.8% (P < 0.05), respectively. An equimolar dose of the cytotoxic radical alone, AN-201, had no significant effects on any of these parameters. The effect of treatment with AN-238 could be nullified by administering 200 μg of the SST analog, RC-160, 15 minutes before the injection of AN-238 (Fig. 2 and Table 1).
In Experiment 3, a single injection of AN-238 at 200 nmol/kg significantly inhibited the growth of ER-negative AN3CA human endometrial carcinoma xenografts, tumor volumes being 56.9% smaller than in controls (P < 0.01). The tumor doubling time also was significantly prolonged (P < 0.05) and tumor weight was decreased by 59.5% (P < 0.05). Equimolar doses of the cytotoxic radical, AN-201, had no significant effects (Fig. 3 and Table 1).
Side Effects and Toxicity
Analog AN-238 at a single dose of 200 nmol/kg caused no significant decrease of the leukocyte count in any of the experiments, whereas radical AN-201 at the same dose significantly (P < 0.05) suppressed the leukocyte count on Day 8 in all 3 experiments (Table 1).
A minor body weight loss of 6.3–10.7%, compared with controls was observed on treatment Day 8 after the injection of AN-201 and AN-238 at 200 nmol/kg. This loss was significant for AN-238 and AN-201 (P < 0.05) in Experiments 1 and 2. However, on Day 15, the treated animals in both groups eventually recovered and their body weights no longer differed from animals in the control groups.
No deaths due to drug-related toxicity occurred in Experiments 1 and 3. In Experiment 2, 4 animals died after treatment with AN-201, whereas only 1 death was observed after injection of AN-238. One mouse each also died in the control groups in Experiments 2 and 3, respectively.
Microscopic examinations of the kidneys showed small changes 5 days after the treatment with AN-238 or AN-201 at 200 nmol/kg compared with controls. Few apoptotic cells, some mononuclear cell infiltration, and a slight increase in mitotic cells were detectable in the tubulus cells with no obvious differences between the two treatment regimens.
Expression of mRNA and Receptor Protein for Somatostatin
RT-PCR analyses demonstrated the expression of mRNA for sst1–5 in HEC-1A, RL-95-2, and AN3CA tumor specimens from control animals. The PCR products were of the expected size of 217 base pair (bp) for sst1, 1104 bp for sst2a, 183 bp for sst3, 278 bp for sst4 and 222 bp for sst5 in all 3 cell lines (Fig. 4). No PCR products were amplified from the negative controls, ruling out the possibility of genomic contamination. The presence of sst2 and sst5 receptor protein in untreated HEC-1A, Rl-95-2 and AN3CA tumor tissue specimens was evaluated by Western blotting using specific antibodies. Specific bands showing 43-kilodalton proteins for both sst2 and sst5 were found in all investigated tumor samples (Fig. 5).
In membranes of HEC-1A tumor specimens from the control group, receptor analyses revealed a single class of high-affinity (Kd of 5.84 nM) binding sites for SST, with a mean Bmax of 232.8 fmol/mg membrane protein (Table 2). Using ligand competition assays, specific high-affinity receptors for SST were found in RL-95-2 tumor specimens with a mean Kd value of 1.32 nM and a mean Bmax value of 540.6 fmol/mg membrane protein (Table 2). Binding sites for SST having a mean Kd value of 4.88 nM and a mean Bmax value of 326.9 fmol/mg membrane protein could also be detected in AN3CA tumor tissue specimens (Table 2).
|Cell line||Kd in nM||Bmax in fmol/mg protein|
|Nude HEC-1A||5.84 ± 0.31||232.8 ± 10.1|
|RL-95-2||1.32 ± 0.07||540.6 ± 36.2|
|AN3CA||4.88 ± 0.39||326.9 ± 26.7|
MDR-1 and MRP-1 and BCRP mRNA expression by Real-Time Polymerase Chain Reaction
mRNA for MDR-1, MRP-1, and BCRP was detected in all 3 cell lines (Fig. 6). The PCR products were of the expected sizes of 95 bp for MDR-1, 127 bp for MRP-1, 140 bp for BCRP, and 140 bp for β-actin (Fig. 6). The E values were 1.989 for MDR-1, 1.999 for MRP-1, 1.987 for BCRP, and 1.997 for β-actin.
In HEC 1-A tumor specimens, treatment with AN-238 and AN-201 caused a 1.8 and a 4.0-fold induction of the MDR-1 gene, respectively. In RL-95-2 tumor specimens, therapy with AN-238 and AN-201 was associated with a 13.7 and a 16.1-fold induction of mRNA for MDR-1. In AN3CA tumor specimens, MDR-1 expression was found to be increased 14.4 and 39.5-fold after treatment with AN-238 and AN-201, respectively. AN-238 and AN-201 did not induce MRP-1 or BCRP to a major extent in any experiment (data not shown).
Endometrial carcinoma does not respond well to chemotherapy. Expression of MDR-1 has been detected in most endometrial tumor specimens as well as in normal endometrial tissue specimens.31–33 Thus, endometrial carcinoma appears to belong to the category of tumors arising in organs that normally overexpress MDR-I, all of which tend to be intrinsically resistant to chemotherapeutic treatment. Chemotherapy targeted to hormone receptors on tumors is designed to produce a more selective and more effective drug delivery to malignant lesions. Therefore, using targeted chemotherapy, a high intratumoral concentration of cytotoxic drugs can be attained, which may overcome the MDR of neoplastic cells. Accordingly, in previous studies with LHRH receptor-positive human endometrial tumors, a strong inhibition of tumor growth was observed after treatment with the cytotoxic LHRH analogs, AN-152 and AN-207, which consist of DOX or 2-pyrrolinoDOX linked to [D-Lys6]-LHRH, respectively.47, 48 The results demonstrated a high efficacy of the targeted LHRH analogs on LHRH receptor-positive tumors, whereas the nontargeted DOX and 2-pyrrolinoDOX (AN-201) had only minor effects.47, 48
Binding sites for a radiolabeled SST analog have been detected in the peritumoral vessels of 4 of 10 endometrial tumor specimens.26 A recent study reported the presence of all 5 sst subtypes in human endometrial tumor specimens, sst2a and sst3 being the most frequently expressed with a percentage of 43% and 39%, respectively.27 An ongoing investigation in our laboratory is focusing on the extensive evaluation of the expression of sst in specimens of human primary and metastatic endometrial tumors, as it is important to evaluate whether the sst subtypes are preserved in metastatic disease. Thus, in the current study, we decided to evaluate the therapeutic efficacy of the cytotoxic SST analog, AN-238, in 3 human endometrial carcinoma models in vivo.
Because the cytotoxic octapeptide analog, AN-238, binds with high affinity to sst2a and sst5, we determined the protein expression of these receptor subtypes on all 3 cell lines by using Western blot analysis. Specific, high-affinity binding sites for SST and mRNA for all five sst subtypes were also detected in these cancer lines as determined by ligand binding assays and RT-PCR analysis. Targeting of AN-238 to SST receptors resulted in a significant inhibition of tumor growth in all 3 models, whereas the cytotoxic radical AN-201 was ineffective. AN-238 was very effective in ER-positive RL-95-2 tumors, as well as in HEC-1A and AN3CA tumors, which do not express receptors for estrogen. Because in a previous study we found that high doses of the “straight” SST octapeptide analog, RC-160, inhibited the growth of HEC-1A endometrial tumors in nude mice,49 in Experiment 1, we included the unconjugated carrier peptide RC-121, which is also an octapeptide SST analog. RC-121 at the single dose of 200 nmol/kg did not significantly suppress the growth of HEC-1A cancer xenografts, indicating that the effect of the same dose of AN-238 is not related to the hormonal activity of the conjugate, but rather to its ability to deliver AN-201 to SST receptors on cancerous cells. In a previous study,49 RC-160 was given daily at the dose of 100 μg per mouse (approximately 5 μmol/kg). To further demonstrate that the action of AN-238 was mediated by SST receptors, mice bearing RL-95-2 xenografts were pretreated with an excess of SST analog RC-160 to block the SST receptors before the administration of AN-238. We found that the antitumor activity of AN-238 was strongly decreased by RC-160, demonstrating that the unoccupied SST receptors are obligatory for targeted therapy with AN-238. We also previously demonstrated that AN-238 selectively kills SST receptor-positive UCI-107 human ovarian tumor cells.50 These studies clearly demonstrate the essential role of SST receptors for targeted therapy with the cytotoxic SST analog, AN-238.
Because the sst subtypes are widely distributed in the kidney, gastrointestinal tract, and other tissue types,11, 51, 52 it was anticipated that therapy with radionuclide or cytotoxic SST analogs may produce some target-specific side effects. However, in early studies with radiolabeled SST analogs, no or only low-grade toxicity was observed in patients at doses resulting in objective responses.16, 17, 53 Severe renal toxicity only appeared in patients treated with very high doses of radionuclide SST analogs and could be prevented by the administration of high amounts of the amino acid lysine before therapy.53 Based on the relatively lipophilic nature of AN-238, renal clearance is not anticipated with this analog. Accordingly, after treatment with AN-238 or AN-201 at the highest dose used in the current study, only small changes were observed in the renal tubulus cells of both groups on Day 5 when toxic side effects were expected to be maximal. The systemic toxicities of the targeted cytotoxic SST analog, AN-238, and its radical, AN-201, were compared with respect to the incidence of deaths, weight loss, and leukocyte suppression. In all 3 experiments, only 1 mouse died after the injection of AN-238, but 4 deaths occurred after treatment with AN-201. Minor body weight loss of 6.3–10.7% occurred in all groups 8 days after treatment with AN-238 or AN-201, but this effect was transient and on Day 15, the animals recovered. The myelotoxicity is usually the most serious side effect and the dose-limiting factor of chemotherapy. In all experiments, AN-201, but not an equivalent dose of AN-238, significantly suppressed the leukocyte count on Day 8. A nonsignificant decrease in the leukocyte count after treatment with 200 nmol/kg of AN-238 was observed in all 3 studies This is probably due to a high esterase activity in the serum of mice, which can partially cleave the ester bond in AN-238 that couples AN-201 to the carrier peptide. As a result, AN-201 could reach a level in the circulation that would cause a moderate decrease in the leukocyte count. It was previously demonstrated in our laboratory that the inhibition of the esterase activity in mice significantly decreases the toxicity of AN-238.54 Because the esterase activity is much lower in humans than in mice,55 the hematotoxicity of AN-238 (due to AN-201) is expected to be reduced in patients. Taken together, the overall toxicity was less pronounced in animals treated with AN-238 compared with its cytotoxic radical.
Changes of the expression of the MDR-1, MRP-1, and BCRP genes were evaluated by real-time PCR in all experiments. AN-238 caused a weaker induction of MDR-1 than its cytotoxic radical, AN-201, in all 3 cancer lines. Similar results were observed in vitro in the HEC-1A endometrial carcinoma line after treatment with the targeted cytotoxic LHRH analog, AN-152.56 Both AN-152 and its radical, DOX, induced surface expression of the MDR-1 gene product, Pgp, but the effect of AN-152 was smaller than that of DOX.56 Thus, the development of chemoresistance may be delayed by targeted chemotherapy. No major induction of mRNA for MRP-1 and BCRP occurred after treatment with AN-238 and AN-201.
In conclusion, the targeted cytotoxic SST analog, AN-238, is more efficacious and less toxic than the nontargeted chemotherapeutic radical, AN-201, and consequently could be considered for the treatment of SST receptor-positive endometrial tumors. Our findings support the merit of clinical trials with AN-238 in patients with advanced or recurrent endometrial carcinomas. AN-238 should be available for clinical trials in the near future.
The authors thank Patricia Armatis, Miriam Ducruet, and Harold Valerio for excellent experimental assistance and Dudley R. Callais for his help with the preparation of the current article.
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