Jose M. Arencibia

HIGH INCIDENCE OF RECEPTORS FOR LUTEINIZING HORMONE- RELEASING HORMONE (LHRH) AND LHRH RECEPTOR GENE EXPRESSION IN HUMAN PROSTATE CANCERS

PURPOSE Agonistic analogs of luteinizing hormone-releasing hormone (LHRH) are widely used for therapy of advanced prostate cancer based upon their ability to suppress testosterone secretion in patients. Various studies also indicate that LHRH analogs might have direct inhibitory effects on prostate tumors mediated by specific LHRH receptors. In this study we investigated the presence and characteristics of receptors for LHRH and their messenger (m) ribonucleic acid (RNA) expression in specimens of human prostate adenocarcinomas and benign prostatic tissue. MATERIALS AND METHODS In vitro ligand competition assays as well as reverse transcriptase polymerase chain reaction (RT-PCR) were performed to investigate the expression of receptors for LHRH in surgical specimens of human prostate cancers and benign prostatic tissue. RESULTS Sixty-nine of 80 (86%) cancers exhibited specific, medium to high-affinity binding for [D-Trp6]LHRH with a dissociation constant (Kd) of 6.55+/-0.4 nM and a maximal binding capacity (Bmax) of 483.6+/-25.4 fmol./mg. membrane protein. Two prostate cancer patients who were treated with the LHRH agonist goserelin prior to prostatectomy did not show tumor LHRH receptors. The expression of mRNA for LHRH receptors was observed in 19 of 22 (86%) prostate cancers. Benign prostatic tissue also displayed LHRH receptor gene expression, but exhibited lower Bmax value. There was a negative correlation (p <0.001) between LHRH receptor binding capacity and cancer grade (Gleason score); higher Gleason scores were associated with significantly lower binding capacity but an increased binding affinity. CONCLUSIONS The expression of specific receptor proteins for LHRH in human prostate cancer provides a rationale for the improvement in methods for therapy of this malignancy based on LHRH analogs.

Growth inhibition of experimental pancreatic cancers and sustained reduction in epidermal growth factor receptors during therapy with hormonal peptide analogs.

Abstract Reduction in receptors for epidermal growth factor (EGF) in cancers appears to be one of the principal mechanisms through which peptide hormone analogs can inhibit tumor growth. In this study, hamsters with nitrosamine-induced pancreatic cancers were treated for 8 weeks with bombesin/gastrin-releasing peptide (GRP) antagonist RC-3095, somatostatin analog RC-160 or the luteinizing hormone-releasing hormone antagonist Cetrorelix, using sustained delivery systems releasing 20, 35 and 20 μg analog/ day respectively. To establish the pattern of changes in the number and affinity of EGF receptors on tumors, groups of animals were sacrificed at regular intervals during therapy. Chronic treatment with RC-3095 or Cetrorelix resulted in an early (day 10) and sustained reduction (71% or 69% respectively) in EGF receptors on pancreatic tumors. In contrast, RC-160 decreased receptor concentration by 60% only after 20 days. Among the histological characteristics of proliferation, the decrease in argyrophilic nucleolar organizer regions, but not apoptotic and mitotic indices, showed a correlation with the fall in EGF receptors. The concentration of the receptors returned to the control level 4 days after cessation of chronic treatment with RC-3095. The effect of single injections of RC-3095, RC-160 and Cetrorelix on EGF receptors was also investigated. RC-160 decreased the number of EGF receptors on pancreatic cancers by 31% 3 h after administration, but the receptors had returned to normal level at 6 h. RC-3095 and Cetrorelix caused a 67% and 59% decline, respectively, in EGF receptors only 6 h after injection and the concentration of receptors remained low for 24 h. Thus, the pattern of down-regulation of EGF receptors in pancreatic cancers appears to depend on the peptide used for therapy. Since the antitumor effect may be the result of the fall in EGF receptors in cancers, information on the time course of changes in these receptors during treatment with these analogs may lead to an improvement in therapeutic regimens.

Substrate-Selective Inhibition of Protein Kinase PDK1 by Small Compounds that Bind to the PIF-Pocket Allosteric Docking Site

The PIF-pocket of AGC protein kinases participates in the physiologic mechanism of regulation by acting as a docking site for substrates and as a switch for the transduction of the conformational changes needed for activation or inhibition. We describe the effects of compounds that bind to the PIF-pocket of PDK1. In vitro, PS210 is a potent activator of PDK1, and the crystal structure of the PDK1-ATP-PS210 complex shows that PS210 stimulates the closure of the kinase domain. However, in cells, the prodrug of PS210 (PS423) acts as a substrate-selective inhibitor of PDK1, inhibiting the phosphorylation and activation of S6K, which requires docking to the PIF-pocket, but not affecting PKB/Akt. This work describes a tool to study the dynamics of PDK1 activity and a potential approach for drug discovery.

Inhibition of growth of ES-2 human ovarian cancers by bombesin antagonist RC-3095, and luteinizing hormone-releasing hormone antagonist Cetrorelix

We evaluated the effects of the bombesin/gastrin-releasing peptide (GRP) antagonist RC-3095, and the luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix, administered singly or in combination, on the growth of human ovarian carcinoma cell line ES-2, xenografted into nude mice. RC-3095 at a dose of 20 microg/day and Cetrorelix (100 microg/day), significantly reduced the volume of ES-2 tumors by 63.0% (P<0.01) and 38.0% (P<0.05) respectively, after 44 days of treatment, as compared with controls. The combination of RC-3095 with Cetrorelix inhibited the growth of ES-2 tumors by 66.2% (P<0.01). Serum levels of LH were significantly decreased in the groups treated with Cetrorelix alone and/or in combination with RC-3095. RT-PCR analyses revealed that the expression of mRNA for receptors of GRP (GRPR/BRS-1) and Neuromedin B (NMBR/BRS-2) on tumors was significantly decreased in all the treated groups. The expression of mRNA for epidermal growth factor receptors (EGFR) on tumors was reduced by 36.5 % (P<0.05) in the animals treated with Cetrorelix and by 72.5% (P<0.05) in the group that received the combination of RC-3095 with Cetrorelix. Our results indicate that the bombesin antagonist RC-3095 and the LH-RH antagonist Cetrorelix inhibit effectively the growth of ES-2 ovarian cancers in nude mice. These antagonists and their combination could be considered for the therapy of patients with ovarian cancer.

Administration of a targeted cytotoxic analog of luteinizing hormone-releasing hormone inhibits growth of estrogen-independent MDA-MB-231 human breast cancers in nude mice

Receptor targeted chemotherapy is less toxic and more effective than conventional chemotherapy. Receptors for luteinizing hormone-releasing hormone (LH-RH) are found in about 50% of human breast cancers. Highly potent cytotoxic radical 2-pyrrolinodoxorubicin (AN-201) was linked to the agonistic analog [D-Lys6]LH-RH to form cytotoxic LH-RH analog AN-207. We evaluated whether AN-207 could be targeted to the hormone-independent MDA-MB-231 human breast cancers. Nude mice bearing MDA-MB-231 tumors were injected i.v. with 250 nmol/kg doses of cytotoxic radical AN-201, cytotoxic LH-RH analog AN-207, the unconjugated mixture of AN-201 and carrier [D-Lys6]LH-RH, [D-Lys6]LH-RH alone and vehicle (control). The growth of MDA-MB-231 tumors in animals given a single dose of AN-207 was inhibited significantly (p=0.01) for 3 weeks after injection, whereas tumors in all the other groups grew steadily. All cytotoxic compounds produced leukopenia, but the strongest lymphocyte suppression was caused by cytotoxic radical AN-201. Three weeks after treatment, the presence of mRNA for LH-RH receptors was demonstrated by RT-PCR in all the groups and radioreceptor assays demonstrated high-affinity binding sites for LH-RH on tumor cell membranes of control animals and those treated with AN-201, the carrier peptide alone or in combination with AN-201. At this time point binding assays did not reveal the expression of membrane proteins in tumors treated with AN-207, but 60 days after administration of AN-207, high affinity LH-RH binding sites were found again in MDA-MB-231 tumors. These results indicate that cytotoxic LH-RH analog AN-207 could be utilized for receptor targeted chemotherapy of breast cancers expressing receptors for LH-RH.

Improving Drug Penetrability with iRGD Leverages the Therapeutic Response to Sorafenib and Doxorubicin in Hepatocellular Carcinoma.

iRGD is a derivative of the integrin-binding peptide RGD, which selectively increases the penetrability of tumor tissue to various coadministered substances in several preclinical models. In this study, we investigated the ability of iRGD to improve the delivery of sorafenib and doxorubicin therapy in hepatocellular carcinoma (HCC) using established mouse models of the disease. A contrast-enhanced MRI method was developed in parallel to assess the in vivo effects of iRGD in this setting. We found that iRGD improved the delivery of marker substances to the tumors of HCC-bearing mice about three-fold without a parallel increase in normal tissues. Control peptides lacking the critical CendR motif had no effect. Similarly, iRGD also selectively increased the signal intensity from tumors in Gd-DTPA-enhanced MRI. In terms of antitumor efficacy, iRGD coadministration significantly augmented the individual inhibitory effects of sorafenib and doxorubicin without increasing systemic toxicity. Overall, our results offered a preclinical proof of concept for the use of iRGD coadministration as a strategy to widen the therapeutic window for HCC chemotherapy, as monitored by Gd-DTPA-enhanced MRI as a noninvasive, clinically applicable method to identify iRGD-reactive tumors.

Effective treatment of experimental ES-2 human ovarian cancers with a cytotoxic analog of luteinizing hormone-releasing hormone AN-207.

The receptors for luteinizing hormone-releasing hormone (LHRH) are found in 80% of human ovarian carcinomas. These receptors can be used for targeted chemotherapy with cytotoxic analogs of LHRH, such as AN-207, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to [D-Lys6]LHRH. We investigated the effects of AN-207 and AN-201 on the in vivo growth of LHRH receptor-positive ES-2 human ovarian cancers. The effects of the treatment on mRNA and protein levels of human epidermal growth factor (EGF) receptors (EGFR and HER-2) in ovarian tumors were determined by RT-PCR and immunoblotting. In Experiment 1, nude mice bearing ES-2 ovarian tumors were injected i.v. with 250 nmol/kg doses of AN-207, AN-201, the carrier [D-Lys6]LHRH, an unconjugated mixture of AN-201 and [D-Lys6]LHRH or vehicle. AN-207 caused a significant (p <0.01) 59.5% inhibition in tumor growth while its components were ineffective. In Experiment 2, mice with large ES-2 tumors were treated with AN-207 or AN-201 at 250 nmol/kg. Again, AN-207, but not AN-201, inhibited tumor growth. In Experiment 3, the site of action of AN-207 was investigated. The blockade of LHRH receptors with Cetrorelix partially suppressed the antitumor effect of AN-207. Treatment with AN-207 significantly (p <0.01) decreased the expression of mRNA for EGFR, and HER-2 by 27 and 34%, respectively, as compared to controls and reduced the receptor protein levels of EGFR and HER-2 by 35 and 36%, respectively (p <0.05). The results indicate that cytotoxic LHRH analog AN-207 could be considered for chemotherapy of ovarian cancers expressing LHRH receptors.

In vitro targeting of a cytotoxic analog of luteinizing hormone-releasing hormone AN-207 to ES-2 human ovarian cancer cells as demonstrated by microsatellite analyses

Targeting of cytotoxic agents represents a modern approach to the treatment of various cancers, that improves the efficacy and reduces peripheral toxicity. Recently we developed a powerful cytotoxic analog of luteinizing hormone-releasing hormone (LHRH), AN-207, designed to be targeted to tumors that express LHRH receptors. This analog consists of the superactive derivative of doxorubicin (DOX), 2-pyrrolino-DOX (AN-201), linked to [D-Lys6]LHRH carrier. In the present study we investigated the cytocidal effects of AN-207 and AN-201 on the LHRH receptor-positive ES-2 ovarian cancer cells. The targeting of AN-207 to ES-2 cells in the presence of LHRH receptor-negative UCI-107 ovarian cancer cells was also evaluated by semi-quantitative polymerase chain reaction (PCR) amplification of microsatellite markers. Ligand competition assays showed a single class of high-affinity and low-capacity binding sites in ES-2 cells with a mean dissociation constant (KD) of 3.93±0.1 nM and a mean maximal binding capacity (Bmax) of 271±26.1 fmol/mg membrane protein. Kinetic assays indicated that AN-207 caused cell death in a concentration- and time-dependent manner in ES-2 cells, but not in UCI-107 cells, while the kinetics of cytotoxic effects of AN-201 were similar in both cell lines. To investigate targeting, ES-2 cells were co-cultured with UCI-107 cells, treated with 10 nM AN-207 or AN-201 for different times and then cultured for 48 h in the absence of cytotoxic agents. Genomic DNA was extracted for microsatellite analyses using different markers. Semi-quantitative analyses of the intensity of the alleles that correspond to each cell line indicated that AN-207 was selectively targeted to ES-2 cells, while AN-201 showed no selectivity for either cell line. These results extend our previous findings that AN-207 can be targeted to ovarian cancers and other tumors that express receptors for LHRH. Cytotoxic analogs of LHRH, such as AN-207, should be considered for treatment of LHRH receptor-positive tumors.

An Allosteric Inhibitor Scaffold Targeting the PIF-Pocket of Atypical Protein Kinase C Isoforms

There is a current and pressing need for improved cancer therapies. The use of small molecule kinase inhibitors and their application in combinatorial regimens represent an approach to personalized targeted cancer therapy. A number of AGC kinases, including atypical Protein Kinase C enzymes (PKCs), are validated drug targets for cancer treatment. Most drug development programs for protein kinases focus on the development of drugs that bind at the ATP-binding site. Alternatively, allosteric drugs have great potential for the development of future innovative drugs. However, the rational development of allosteric drugs poses important challenges because the compounds not only must bind to a given site but also must stabilize forms of the protein with a desired effect at a distant site. Here we describe the development of a new class of compounds targeting a regulatory site (PIF-pocket) present in the kinase domain and provide biochemical and crystallographic data showing that these compounds allosterically inhibit the activity of atypical PKCs. PS432, a representative compound, decreased the rate of proliferation of non-small cell lung cancer cells more potently than aurothiomalate, an atypical PKCι inhibitor currently under evaluation in clinical trials, and significantly reduced tumor growth without side effects in a mouse xenograft model. The druglike chemical class provides ample possibilities for the synthesis of derivative compounds, with the potential to allosterically modulate the activity of atypical PKCs and other kinases.