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Section of Experimental Medicine,3 Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112
Endocrine Polypeptide and Cancer Institute,4 Veterans Affairs Medical Center, New Orleans, Louisiana 70112
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONLHRH, LHRH RECEPTORS, AND...SIDE EFFECTSCONCLUSIONSREFERENCES |
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breast, ovarian, endometrial, and prostate cancers, cytotoxic LHRH analogs, doxorubicin, gonadotropin-releasing hormone, gonadotropin-releasing hormone receptor, mammary glands, ovary, prostate, tumor targeting
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONLHRH, LHRH RECEPTORS, AND...SIDE EFFECTSCONCLUSIONSREFERENCES |
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LHRH, LHRH RECEPTORS, AND CANCER |
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TOPABSTRACTINTRODUCTIONLHRH, LHRH RECEPTORS, AND...SIDE EFFECTSCONCLUSIONSREFERENCES |
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Because sex steroids have been implicated in the development of breast and prostate cancers, studies were initiated in patients for the treatment of these malignancies with LHRH agonists. Based on these clinical studies, hormonal therapy with LHRH agonists was approved for the treatment of sex-steroid-dependent conditions, including estrogen-dependent breast cancers and androgen-dependent prostate carcinoma [9]. Although the basic mechanism of action of LHRH analogs on cancerous cells was initially assumed to be indirect, through the suppression of sex steroids, antiproliferative action of these peptide hormones on human breast cancer cell lines in vitro suggested an additional, direct effect, mediated by tumoral LHRH receptors [10–12].
Extensive investigation of the expression of LHRH receptors on human cancer cell lines and tumor specimens has been performed worldwide during the past 2 decades [9]. Although even a few years ago many scientists were skeptical about the expression of LHRH receptors on various cancers, including prostate, ovarian, and endometrial cancers, more and more studies were published confirming the appearance of LHRH receptors on malignant tissues. The binding sites for LHRH on breast cancers were first reported by Miller et al. [10] and Eidne et al. [11] in 1985. Subsequently, Fekete et al. [13] found that 52% of human breast cancer specimens have high-affinity binding sites for the LHRH superagonist [D-Trp6]LHRH (Decapeptyl) (Table 1). Baumann et al. [14] tested 235 human breast cancer specimens, and found binding sites for LHRH on 121 samples (51.5%). The incidence of LHRH receptors in specimens of human epithelial ovarian cancers was found to be near 80% in three independent studies [15–17]. Similar results were obtained with endometrial carcinoma specimens [17–19]. In an early study, Qayum showed that 86% of prostate cancer specimens removed by surgical resection expressed high-affinity LHRH receptors [20]. This observation was confirmed in our laboratory in 80 prostatic carcinoma specimens [21].
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In a recent study by Tieva et al. [22], 13 of 13 biopsy specimens of prostatic cancer were found to express mRNA for LHRH receptor. The authors localized the receptor protein by immunohistochemistry in cancerous epithelial cells. In yet another recent study, Straub et al. demonstrated that 100% of hormone refractory prostate cancer specimens express mRNA for LHRH receptor [23]. In addition to breast, ovarian, endometrial, and prostate cancers, which can be affected by the pituitary/gonadal axis, cancer specimens or cell lines (or both) from some nonreproductive organs were also found to express receptors for LHRH (Table 1), indicating a broad connection between cancerous transformation and LHRH receptor expression. Such cancers include oral, and laryngeal cancers [24], renal cell carcinomas [25], brain tumors [26], melanomas [27], liver cancers [28], ductal pancreatic carcinomas [29, 30], and adenocarcinomas of the colon [31]. The mechanisms involved in the binding to pituitary and tumoral LHRH receptors, the regulation and active states of the receptors, and the role of regulators of G-protein signaling as well as the intercellular responses that follow the binding were previously expertly reviewed by Conn [32] and Castro et al. [33], Stojilkovic and Catt [34], Millar and Pawson [35], a group of researchers led by McArdle [36–38], and Naor and Childs [39]. However, classical signal transduction mechanisms of LHRH receptors on tumors appear to be different from those for the pituitary [40]. We have also reviewed some aspects of pituitary and tumoral LHRH receptors and the effects of various treatments on receptor levels [41].
It had been shown that the expression of LHRH receptors in tumors is associated with activation of the human epidermal growth factor receptor (EGFR). Thus, an activated EGFR can phosphorylate a 60-kDa protein that corresponds to the LHRH receptor [42–44]. The function of the LHRH receptors in tumors is not well understood, but studies indicate an autocrine/paracrine LHRH-LHRH-receptor loop in human breast [10, 11], prostatic [45], ovarian [46], and melanoma cancer cell lines [27]. Cloning of the mRNA for LHRH receptor in breast and ovarian cancers demonstrated that their nucleotide sequence is identical with that of the pituitary receptor [47]. However, some studies with adenocarcinoma of the colon indicate that an LHRH binding site other than the pituitary LHRH-I receptor may be involved in binding of LHRH analogs [31].
In contrast to tumor cells, LHRH receptors are expressed only in low levels in cells of healthy organs, including the prostate, testes, and ovary, but are not present on most normal tissues [22, 29, 48]. These findings make LHRH receptors a distinct target for cancer therapy. In fact, numerous conjugates consisting of an LHRH vector molecule linked to an antitumor agent have been synthesized and tested in experimental oncological models in vitro and in vivo, and antibodies to LHRH have been also used [1, 31, 49–55].
LHRH Analogs Linked to Anticancer Agents
Soon after the finding of LHRH receptors on breast cancer cells in the mid-1980s, our group designed and synthesized the first series of cytotoxic LHRH conjugates [3, 7]. These hybrid molecules were based on agonistic and antagonistic analogs of LHRH, linked to cytotoxic moieties such as the antimetabolite methotrexate, DNA alkylating agent cisplatin, and the DNA intercalating agent DOX [3, 7]. Some analogs carried complexes of heavy metal ions such as Cu(II) and Ni(II). As an LHRH agonist carrier, we selected [D-Lys6]LHRH, because it was well known that this peptide can be modified at the epsilon amino side chain of its D-Lys6 moiety by large molecules without loss of its ability to bind to LHRH receptors. In fact, attachment of a bulky hydrophobic cytotoxic compound to the D-Lys6 side chain, such as 2-hydroxymethylanthraquinone produced a cytotoxic LHRH agonist, designated T-98, with five times higher LH-releasing activity than [D-Lys6]LHRH in a perifused rat pituitary system [56].
T-98 and another early LHRH agonist analog with methotrexate, coded AJ-04, showed a somewhat increased in vivo antitumor activity in rat Dunning R-3327-H prostatic carcinoma compared to the carrier peptide, [D-Lys6]LHRH, but the hormonal and cytotoxic effects of the conjugates could not be separated in these studies [57]. Another early cytotoxic LHRH conjugate, coded T-107, consists of [D-Lys6]LHRH linked through a glutaric acid spacer to the daunosamine nitrogen moiety of DOX [41]. Unfortunately, this analog had no effect on the growth of MXT breast cancers in vivo, because acylation of the daunosamine nitrogen functionality in DOX significantly reduced its antiproliferative activity [58]. To form cytotoxic LHRH conjugates containing DOX with preserved cytotoxicity, we linked DOX through its 14-O-hemiglutarate to [D-Lys6]LHRH (AN-152), and also to LHRH antagonist sequences [6, 7]. These analogs display high binding affinity to receptors for LHRH on rat pituitary and human breast cancer specimens, and fully preserve the cytotoxic activity of the DOX moiety [6, 59].
Based on reports about daunosamine-modified derivatives of DOX that are about 100 times more potent in vitro than DOX, we set out to further improve on the potential of targeted therapy by synthesizing a series of daunosamine-modified derivatives of DOX that may be suitable for covalent linkage to an LHRH analog [7]. Our efforts led to the development of a highly active DOX derivative, called 2-pyrrolino-DOX (AN-201), which is 500–1000 times more potent than DOX in vitro [60]. Intensely potent derivatives of DOX reported by others, including cyanomorpholino-DOX, morpholino-DOX, methoxymorpholino-DOX, and diacetoxypent-1-yl-DOX, showed similar cytotoxic activities to AN-201 in various cancer cell lines [6, 7]. A common feature of these analogs is that they all have a masked, latent aldehyde functionality appended to the daunosamine moiety of DOX, which can alkylate a guanine base at the intercalation site of DOX [6, 7, 61]. These DOX derivatives were found to be active against DOX-resistant tumors. In addition, although the main dose-limiting toxicity of DOX is cardiomyopathy, these highly active DOX derivatives cause no toxicity to the heart, possibly due to their low maximum tolerated doses (MTDs). Their dose-limiting toxicity appears to be myelosuppression, as these agents can kill all types of rapidly proliferating cells [7].
After the synthetic procedure for the conversion of DOX to 2-pyrrolino-DOX was established [60], we prepared superactive cytotoxic LHRH conjugate AN-207 from AN-152 by the same synthetic method [6, 7]. Cytotoxic LHRH antagonist analogs were also synthesized and evaluated in assays in vitro [6]. The selection for further development of cytotoxic LHRH agonists AN-152 and AN-207 over their LHRH antagonist counterparts was based on a comprehensive study with cytotoxic LHRH agonist and cytotoxic LHRH antagonist analogs containing a derivative of DOX called 3'-deamino-3'-pyrrolidin-1-yl-DOX [5]. In that study, the cytotoxic LHRH antagonist was much more toxic and less active than its agonist counterpart. Based on these results, we designed a series of preclinical studies to investigate the efficacy and the toxicity profiles of cytotoxic LHRH agonists AN-152 containing DOX and AN-207 with 2-pyrrolino-DOX [1, 3–5, 7, 9]. AN-207 should be more efficacious than AN-152 on tumors containing lower levels of LHRH receptors.
Before presenting a detailed overview of preclinical results with these analogs, it is important to cite the studies of several other research groups that are also working on exploiting the high tumor-specific expression of LHRH receptors [31, 44–55]. Various approaches, including toxins conjugated to LHRH antibodies, have also been used for targeting LHRH receptors on tumors [31, 49–55]. Jacobs et al. [52] demonstrated that an antiserum to LHRH inhibits the growth of xenografts of MCF-7 breast cancer cell line in nude mice. It was also shown that bovine RNase A conjugated to LHRH can be targeted to prostate and breast cancer cells, producing a dose-dependent inhibition of tumor growth [51]. LHRH and its analogs have also been fused with or linked to bacterial and plant toxins, including Pseudomonas exotoxin fragments, and pokeweed antiviral protein to target and specifically kill cancerous cells [31, 50, 53–55]. Schlick et al. [55] and Qi et al. [50] reported that a conjugated or recombinant fusion protein composed of pokeweed antiviral protein linked to an analog of LHRH can inhibit prostatic, breast, ovarian, and endometrial cancer cells expressing LHRH receptor. The suppression of growth in vitro and in vivo of a colon adenocarcinoma cell line xenografted into nude mice by chimeric protein composed of LHRH analog and a modified form of Pseudomonas exotoxin was reported by Ben-Yehudah et al. [53, 54]. To avoid the high immunogenic reactions caused by bacterial proteins, human proapoptotic BCL-2 proteins BIK, BAK, and BAX, as well as a downstream protein of the apoptotic cascade, the caspase activated DNase DFF40, were also coupled to LHRH by protein fusion [31]. These chimeric proteins induced apoptosis and inhibited growth of colorectal and renal cancers [31]. Leuschner et al. [49] produced LHRH fusion polypeptides containing the so-called lytic peptides hecate and phor-14. These lytic peptides can dissolve cell membranes and indiscriminately kill all kinds of cells if not coupled to a vector delivering them to cancers. All these LHRH-based targeting approaches produced compounds that show LHRH-receptor-mediated anticancer activity either in vitro or in vivo (or both) with improved efficacy and reduced toxicity compared to the nontargeted antiproliferative agent they contain [31, 49, 50–55].
Breast Cancers
Breast cancer is the most common malignancy in women and the second leading cause of cancer-related deaths [9, 62]. Although early detection and effective treatment modalities for estrogen-dependent breast cancers, including hormonal manipulations with tamoxifen or raloxifen and oophorectomy improved the overall cure rate, the prognosis for estrogen-independent breast cancers is poor [9]. The primary treatment modality of this phenotype, which constitutes more than 60% of all cases, is cytoreductive surgery followed by adjuvant chemotherapy, radiotherapy, or both [62]. In about 25% of these cases, the EGFR-2 is overexpressed, and despite the introduction of Herceptin, a monoclonal antibody to this receptor protein, the 5-yr survival rate is the lowest in this subclass of patients [62]. Chemotherapy is also hindered by intrinsic or acquired resistance of breast cancer cells. Because more than 50% of human breast cancers express receptors for LHRH, a targeted chemotherapy with cytotoxic LHRH analogs AN-152 or AN-207 may help overcome these drawbacks [1, 3–5, 7, 9, 13, 14]. AN-152 and AN-207 have high-affinity binding to human breast cancer specimens characterized by IC50 values of 7.45 ± 0.61 and 6.15 ± 0.56 nM, respectively [59]. These IC50 values were determined by using radioiodinated [D-Trp6]LHRH, and represent the concentrations of the cytotoxic analogs required to displace 50% of the radiotracer ligand.
The LHRH receptor-mediated entry of AN-152 into MCF-7 human breast cancer cells was studied by coupling a two-photon-emitting fluorophore to the daunosamine moiety of DOX in AN-152 to form AN-152:C625. Real-time optical tracking of this low-energy emitting probe in living MCF-7 cells showed that its binding to LHRH receptors is followed by internalization and appearance in the nucleus within 20 min [63]. The expression of LHRH receptors, and the intensity of internalization as well as the cytotoxic activity of AN-152, could be increased by a pretreatment with epidermal growth factor [43]. No binding and internalization of AN-152:C625 was observed with LHRH receptor-negative UCI-107 human ovarian cancer cells, demonstrating that LHRH receptors are a prerequisite for the entry of AN-152 into cells [43].
The in vivo efficacy of AN-152 and AN-207 was compared to their respective nontargeted cytotoxic radicals, DOX and AN-201 in estrogen-independent MXT mouse mammary carcinoma [58]. Because this type of tumor grows at a very high proliferation rate, we started the treatment 1 day after transplantation of the tumors. AN-152 and AN-207 produced a 89%–93% tumor growth inhibition at their MTDs, whereas the nontargeted cytotoxic radicals DOX and AN-201 were highly toxic at the same doses [58]. In these experiments, AN-207 was used at about 100 times lower doses than AN-152 to obtain similar antitumor effects. In another study with MXT cancers, tumors of various sizes were treated with the same analogs. Even large tumors of more than 2000 mm3 responded to treatment with AN-207, but AN-201, AN-152, and DOX had no effects at such an advanced stage of tumor growth [64].
The efficacy of targeted therapy with AN-207 was also assessed in the LHRH receptor-positive, estrogen-independent, DOX-resistant MX-1 human mammary carcinomas in nude mice [65]. In this study, a single injection of AN-207 at a 250 nmol/kg dose cured 10 of 10 mice without apparent toxicity. In contrast, AN-201 killed one animal and eradicated MX-1 cancers only in 1 of 10 mice. To test whether targeting DOX to LHRH receptors on MX-1 cancers can overcome the DOX resistance of this tumor line, we treated tumor-bearing nude mice with five consecutive injections of AN-152 or DOX at doses equivalent to 3 mg/ kg DOX [62]. Although DOX had no effect on the growth of MX-1 tumors, AN-152 showed a significant suppression of tumor growth as assessed by differences in tumor volume, weight, and tumor doubling time compared to controls. The expression of mRNAs for EGFR-2 and EGFR-3, and also their protein levels, were significantly down-regulated by treatment with AN-152, but not by DOX [62]. These findings are especially important in view of the fact that patients with high levels of EGFR-2 have the poorest prognosis, and that this subpopulation of patients is presently treated with Herceptin, a monoclonal antibody to EGFR-2, which blocks the EGFR-2-mediated mitogenic signaling [1].
Advantages of LHRH receptor-targeted therapy with AN-207 were also demonstrated in estrogen-independent MDA-MB-231 human breast cancers in nude mice [66]. A single injection of AN-207 at a dose of 250 nmol/kg caused an initial regression of tumors in 8 of 13 mice. In contrast, tumors grew steadily in animals treated with AN-201 at the same dose, or with a mixture of AN-201 and the carrier [D-Lys6]LHRH. The effects of AN-207 and AN-201 were also compared in the estrogen-independent MDA-MB-435 human breast cancers grown orthotopically in the mammary fat pad of nude mice [67]. A tumor growth suppression of more than 70% by AN-207 was accompanied by a complete inhibition of metastatic spread of the cancers to lymph nodes. AN-201 had no significant effects on tumor volume or weight, and three of eight mice developed lymphatic metastases.
Epithelial Ovarian Cancers
Epithelial ovarian cancer is the fourth most frequent cause of cancer-related deaths in women, primarily because it is usually diagnosed at a late stage [9]. Cytoreductive surgery combined with paclitaxel and platinum-based chemotherapy initially induces a high rate of response, but most patients experience a recurrence of the disease. The presence of LHRH receptors on more than 80% of ovarian cancer specimens makes it possible to target ovarian cancers with cytotoxic LHRH analogs such as AN-152 and AN-207 [1, 3–5, 7, 9, 15, 16].
In an early study with LHRH-receptor-positive OV-1063 human epithelial ovarian cancers in nude mice, we administered AN-152 and DOX i.p. at a single dose of 20 µmol/ kg, equivalent to 12 mg/kg DOX [68]. Although AN-152 caused a strong antitumor effect with no severe toxicity, DOX killed six of nine animals without inhibiting tumor growth. At a much higher dose, 35 µmol/kg, corresponding to 20.3 mg/kg DOX, AN-152 caused an even stronger (>90%) tumor growth inhibition, but killed two of nine mice. In a second experiment, AN-152 was given twice at a dose of 20 µmol/kg, equivalent to 12 mg/kg DOX, or once at 30 µmol/kg. In this experiment, we measured the binding characteristics of EGFR, and found that after treatment with AN-152, the number of EGFRs was significantly down-regulated. Because an overexpression of EGFR is a bad prognostic indicator in ovarian carcinoma, its down-regulation by AN-152 is very promising for patients with this disease. In the same study, we also treated LHRH receptor-negative UCI-107 human epithelial ovarian cancers in nude mice to evaluate the importance of LHRH receptor expression in targeted chemotherapy with AN-152. Neither AN-152 nor DOX affected the growth of UCI-107 cancers at doses of 20 µmol/kg, equivalent to 12 mg/kg DOX, demonstrating that the presence of LHRH receptors on tumors is a prerequisite for effective treatment with AN-152 [68]. Similar results were obtained with AN-207 in these two ovarian cancer models, but at about 100 times lower doses [69].
The efficacy of LHRH receptor targeting with AN-152 and AN-207 was also tested in the LHRH receptor-positive ES-2 human ovarian cancers in nude mice [70, 71]. A single injection of AN-152 to tumor-bearing mice at a dose of 17 µmol/kg, equivalent to 10 mg/kg DOX, had a significant tumor growth inhibitory effect accompanied by a significant decrease in the levels of mRNA for EGFR-2, vascular endothelial growth factor, and the oncogenes FOS and JUN [70]. In contrast, DOX produced no growth inhibition at the same dose, and no significant effects on these molecular markers could be measured after therapy. Similarly to AN-152, a single i.v. injection of AN-207 at a dose of 250 nmol/kg significantly inhibited the growth of ES-2 tumors in nude mice, and reduced the expression of EGFR and EGFR-2 on both the mRNA and the protein levels, whereas the nontargeted cytotoxic radical AN-201 had no significant effects on tumor growth [71]. The ability of AN-207 to selectively kill LHRH receptor-expressing cancer cells was demonstrated in vitro by a microsatellite analysis of a mixed population of LHRH receptor-positive ES-2 cells and LHRH receptor-negative UCI-107 cells [72].
The efficacy of cytotoxic LHRH analog AN-152 and DOX was compared in the LHRH receptor-positive NIH: OVCAR-3 human epithelial ovarian cancer model in nude mice [48]. One week after a single i.v. injection of AN-152 at a dose of 15 µmol/kg, equivalent to 8.7 mg/kg DOX, a shrinkage of the initial tumor size by about 30% was observed. A higher dose of AN-152, 35 µmol/kg, equivalent to 20.3 mg/kg DOX, was not any more effective, probably due to a saturation of the LHRH receptors by a dose of 8.7 mg/kg. No toxicity-related deaths occurred in these experiments. When the nontargeted cytotoxic agent DOX was used at a 15 µmol/kg dose (8.7 mg/kg), it also produced a significant inhibition of tumor growth, but no reduction of the initial tumor volume was achieved [48]. DOX given at a dose of 35 µmol/kg (20.3 mg/kg) killed three of five mice, suggesting again that systemic chemotherapy is more toxic than LHRH-based targeted chemotherapy. When nude mice xenografted with LHRH receptor-negative SKOV-3 tumors were treated with AN-152 at a dose of 15 µmol/kg, equivalent to 8.7 mg/kg DOX, no antitumor effects were observed, supporting the view that the expression of LHRH receptors on tumors are essential for an effective therapy with AN-152 [48].
Endometrial Cancers
Endometrial carcinoma is the most common neoplasm of the female genital tract, but the 5-yr survival rate is relatively high due to early detection of the disease [73]. However, in patients with recurrent endometrial cancer, the overall survival rate is poor. The presence of LHRH receptors on more than 80% of human endometrial carcinoma specimens provides a rationale for the use of targeted cytotoxic LHRH analogs to treat patients with advanced cancers [1, 3–5, 7, 9, 18, 19].
LHRH receptor-mediated internalization of AN-152 by HEC-1A and Ishikawa human endometrial cancer cells was demonstrated in vitro by confocal laser scanning microscopy using the autofluorescence of the DOX moiety in AN-152 [74]. Dynamic experiments with living HEC-1A cells showed that although DOX appeared in the nuclei of cells within 5 min after exposure, at the same time, AN-152 accumulated in cytoplasmic granules. After 10 min of exposure, AN-152 concentrated in bigger granules near the nucleus, and in 15 min, all the fluorescent signal of AN-152 was located within the nucleus. This receptor-mediated entry of AN-152 could be blocked by LHRH agonist Decapeptyl ([D-Trp6]LHRH). The necessity of LHRH receptors for cellular entry of AN-152 was further shown by the fact that no cellular uptake of AN-152 could be observed by LHRH receptor-negative SKOV-3 human ovarian cancer cells [74]. DOX entered both cell types indiscriminately.
The efficacy of LHRH receptor-based targeted therapy of endometrial cancers was demonstrated in HEC-1A, HEC-1B, and RL-95-2 human endometrial cancers xenografted into nude mice [48, 73]. One week after a single injection of AN-152 at a dose of 15 µmol/kg, equivalent to 8.7 mg/kg DOX, the size of initial HEC-1B tumors was reduced by about 25% (P < 0.05 vs. control) [48]. DOX at the same dose was significantly less effective than AN-152 (NS vs. control). Although DOX could slow down the tumor growth rate, it did not cause a shrinkage of the initial tumors. At the dose of 35 µmol/kg, equivalent to 20.3 mg/ kg DOX, AN-152 had similar effects to those obtained at the lower dose, which may be due to a saturation of LHRH receptors. All five mice survived the treatment with this high-dose chemotherapy. However, DOX at the same dose killed four of five mice, again indicating a higher toxicity of systemic chemotherapy compared to LHRH-based targeted therapy [48]. In another study, a single injection of AN-152 at a dose of 17 µmol/kg, equivalent to 10 mg/kg DOX, significantly inhibited the growth of xenografts of HEC-1A cancers in nude mice, but 2 of 10 animals died before the end of the observation period [73]. DOX at the same dose was toxic, killing four of nine mice, and had no significant tumor inhibitory effects. In the same study, targeted cytotoxic LHRH analog AN-207 also exerted a significant tumor growth inhibition of HEC-1A and RL-95-2 human endometrial cancer xenografts in nude mice, whereas the nontargeted cytotoxic radical, AN-201, had no significant effects [73]. Recently, in a collaboration, we were able to show that internalization of cytotoxic analog AN-152 of LHRH as monitored by confocal laser scanning microscopy, induces apoptosis in human endometrial and ovarian cancer cell lines [75].
Prostate Cancers
Prostate cancer is the most common noncutaneous malignancy in men [76]. In spite of major improvements in diagnosis, including screening for serum prostate specific antigen (PSA), many prostate cancers are detected at an advanced stage with no possibility of cure by radical prostatectomy [9, 76]. Because about 70% of prostate cancers are androgen-dependent at the time of diagnosis, the preferred treatment is androgen ablation. However, after an initial remission, most patients relapse and develop androgen-independent prostate cancer, for which the prognosis is very poor [9, 76]. Clinical trials with conventional chemotherapy regimens performed in patients with hormone-refractory prostate cancer indicate that overall response rates are very low and associated with high general toxicity [76]. A local delivery of chemotherapeutic agents may help improve the efficacy and lower the toxicity of systemic chemotherapy [76].
Because LHRH receptors are expressed on 86% of human prostate cancer specimens, we tested our targeted cytotoxic LHRH conjugates in various experimental models of prostatic carcinoma [1, 3–5, 7, 9, 20, 21, 76]. In an early study, we evaluated the efficacy of AN-207 on the LHRH receptor-positive androgen-dependent rat Dunning R-3327-H transplantable prostate tumors [77]. We found that even very large initial tumors, measuring more than 8500 mm3 in size, regressed by about 50% after three consecutive i.p. injections of AN-207 at 50 nmol/kg doses. In contrast, the nontargeted cytotoxic radical, AN-201 killed all rats after the second i.p. injection of 50 nmol/kg. In a second experiment, AN-201 was given three times at a 25 nmol/kg dose, which was still toxic, killing 6 of 10 rats, and exerting only a 57% inhibition of tumor volume, but no tumor regression in the 4 surviving rats [77]. AN-207 also had a strong growth inhibitory effect in the androgen-dependent transplantable PC-82 human prostate tumor line in nude mice [78]. AN-207 at a single dose of 200 nmol/kg caused an initial shrinkage of tumors and a 68% reduction in tumor volume at the end of the study. One of eight mice died due to toxicity. Cytotoxic radical AN-201 had no significant effects and was toxic at the same dose, killing three of eight mice [78].
A lower toxicity and higher efficacy of targeted cytotoxic LHRH analog AN-207 compared to the nontargeted radical AN-201 was also observed in the LHRH receptor-positive MDA-PCa-2b prostate cancers xenografted into nude mice [79]. This cancer cell line originated from a metastatic lesion in the thoracic vertebrae of a patient with progressive disease despite previous orchiectomy, and treatment with DOX and suramine [79]. The MDA-PCa-2b prostate cancer cell line produces PSA, and is considered as a representative experimental model of advanced prostatic carcinoma that acquired androgen-independent growth properties [79]. AN-207 at a cumulative dose of 350 nmol/kg caused a significant suppression of tumor growth without apparent toxicity, while AN-201 had no significant effects and killed three of seven mice after a single injection of 200 nmol/ kg. Serum PSA levels were inhibited significantly only by AN-207 at the end of the observation period [79]. The effects of DOX-containing cytotoxic LHRH analog AN-152 and DOX were also compared in the MDA-PCa-2b model [80]. Five consecutive injections of AN-152 at a dose of 7 µmol/kg, equivalent to 4 mg/kg DOX, resulted in a significant inhibition of tumor growth compared to controls as measured by changes in tumor volume, weight, and tumor doubling time. AN-152 was also significantly more effective than DOX (P < 0.05), which had only nonsignificant effects.
AN-152 and DOX have likewise been tested in androgen-independent C4-2 cancers, which were derived from the androgen-dependent LNCaP prostate cancer cell line [80]. C4-2 cells were grown intraosseously in the tibiae of nude mice and treated with four consecutive injections of AN-152 or DOX at a dose equivalent to 4 mg/kg DOX. AN-152 significantly suppressed the production of PSA by C4-2 cancer cells and significantly increased the apoptotic index in tumors compared to controls. In contrast, DOX had no significant effects on either parameter [80]. To achieve an increase in apoptosis in prostate cancers is of special relevance, because in general, a reduced rate of apoptosis is characteristic of prostatic carcinomas [81].
Other LHRH Receptor-Expressing Cancers
Based on recent findings indicating that LHRH receptors are expressed on a variety of cancers that are not related to the pituitary/gonadal system, we started investigating the efficacy of cytotoxic LHRH analog therapy in these cancers in nude mice. Thus, initial studies showed promising results in experimental models of human malignant melanomas, renal cell carcinomas, colon cancers, and hepatocellular carcinomas. Studies are also in progress in non-Hodgkin lymphomas. A recent in vitro study indicates that oral and laryngeal cancers that express LHRH receptors have an improved response to treatment with AN-152 compared to DOX [24].
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SIDE EFFECTS |
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TOPABSTRACTINTRODUCTIONLHRH, LHRH RECEPTORS, AND...SIDE EFFECTSCONCLUSIONSREFERENCES |
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To evaluate the effects of our cytotoxic LHRH analog AN-207 on the pituitary, rats were treated with a high dose of the compound. Our results indicate that AN-207 has only a transient effect specifically on pituitary gonadotroph cells, but not on GH-producing somatotroph or prolactin-producing cells [83]. In contrast, the nontargeted cytotoxic moiety, AN-201, had a transient toxicity on all cell types. The finding that no permanent damage to the pituitary was caused by AN-207 can be explained by considering the general mechanism of cytotoxicity of AN-207. The cytotoxic radical AN-201, which is linked to [D-Lys6]LHRH through a glutaric acid ester bond to form AN-207, can be released by carboxyl esterase (CE) enzymes (EC.3.1.1.1), which are ubiquitous [84]. Therefore, it is assumed that most of the cytotoxic effects of AN-207 are caused by AN-201 after it is hydrolyzed by these enzymes inside target tissue. Because AN-201 is a DNA-intercalating agent, it primarily kills rapidly proliferating cells, including tumor cells and cells of the bone marrow, but not pituitary cells, which are known to have a relatively long cell cycle [7, 83].
In general, our results in rodents suggest that the toxicity of AN-207 or AN-152 is due to the relatively high CE activity in the serum of these animals, which causes a partial release of AN-201 or DOX, respectively, from the conjugates before they can reach their LHRH receptor-positive target tissue [84]. Accordingly, AN-207 or AN-152 always caused milder side effects than AN-201 or DOX at the same dose. In most cases, these toxicities were manifested as low white blood cell count, or a loss of body weight (or both) [1, 3–5, 7, 9]. Because the half-life of AN-207 and AN-152 in human serum is about 2 h, much longer than the 20 min in nude mice, the toxicity related to a partial release of their respective cytotoxic radical in the blood stream is anticipated to be much lower in patients than in mice [84]. In fact, in vivo inhibition of CE in nude mice resulted in a 50% higher tolerance to AN-207 by the animals, supporting this theory [84].
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONLHRH, LHRH RECEPTORS, AND...SIDE EFFECTSCONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 504 566 1625; aschally{at}tulane.edu
Received: 5 May 2005.
First decision: 2 June 2005.
Accepted: 15 July 2005.
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TOPABSTRACTINTRODUCTIONLHRH, LHRH RECEPTORS, AND...SIDE EFFECTSCONCLUSIONSREFERENCES |
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