Artur Plonowski

Hypothalamic hormones and cancer.

The use of peptide analogs for the therapy of various cancers is reviewed. Inhibition of the pituitary-gonadal axis forms the basis for oncological applications of luteinizing hormone-releasing hormone (LH-RH) agonists and antagonists, but direct effects on tumors may also play a role. Analogs of somatostatin are likewise used for treatment of various tumors. Radiolabeled somatostatin analogs have been successfully applied for the localization of tumors expressing somatostatin receptors. Studies on the role of tumoral LH-RH, growth hormone-releasing hormone (GH-RH), and bombesin/GRP and their receptors in the proliferation of various tumors are summarized, but the complete elucidation of all the mechanisms involved will require much additional work. Human tumors producing hypothalamic hormones are also discussed. Treatment of many cancers remains a major challenge, but new therapeutic modalities are being developed based on antagonists of GH-RH and bombesin, which inhibit growth factors or their receptors. Other approaches consist of the use of cytotoxic analogs of LH-RH, bombesin, and somatostatin, which can be targeted to receptors for these peptides in various cancers and their metastases. These new classes of peptide analogs should lead to a more effective treatment for various cancers.

Peptide analogs in the therapy of prostate cancer

The use of peptide analogs in the therapy of prostate cancer is reviewed. The preferred primary treatment of advanced androgen‐dependent prostate cancer is presently based on the use of depot preparations of LH‐RH agonists. This treatment is likewise recommended in patients with rising PSA levels after surgery or radiotherapy. LH‐RH agonists with or without antiandrogens can be also utilized prior to or following various local treatments in patients with clinically localized prostate cancer and at high risk for disease recurrence. LH‐RH antagonists like Cetrorelix are in clinical trials. However, most patients with advanced prostatic carcinoma treated by any modality of androgen deprivation eventually relapse. Treatment of relapsed androgen‐independent prostate cancer remains a major challenge, but new therapeutic modalities are being developed based on antagonists of growth hormone‐releasing hormone (GH‐RH) and bombesin, which inhibit growth factors or their receptors. Another approach consists of cytotoxic analogs of LH‐RH, bombesin, and somatostatin containing doxorubicin or 2‐pyrrolinodoxorubicin, which can be targeted to receptors for these peptides found in prostate cancers and their metastases. These cytotoxic analogs inhibit growth of experimental androgen‐dependent or ‐independent prostate cancers and reduce the incidence of metastases. A rational therapy with peptide analogs could be selected on the basis of receptors present in biopsy samples. The approaches based on peptide analogs should result in a more effective treatment for prostate cancer. Prostate 45:158–166, 2000.

Potentiation of the inhibitory effect of growth hormone-releasing hormone antagonists on PC-3 human prostate cancer by bombesin antagonists indicative of interference with both IGF and EGF pathways

In view of the involvement of various neuropeptides and growth factors in the progression of androgen‐independent prostate cancer, we investigated the effects of antagonists of growth hormone‐releasing hormone (GHRH) alone or in combination with an antagonist of bombesin/gastrin‐releasing peptide (BN/GRP) on PC‐3 human prostate cancers.

in vivo inhibition of PC-3 human androgen-independent prostate cancer by a targeted cytotoxic bombesin analogue, AN-215

The effectiveness of chemotherapy targeted to bombesin (BN) receptors was evaluated in nude mice bearing PC‐3 human androgen‐independent prostate cancers. Cytotoxic BN analogue AN‐215, consisting of 2‐pyrrolinodoxorubicin (AN‐201) linked to BN‐like carrier peptide RC‐3094, was injected i.v. at 150 nmol/kg on days 1, 11 and 21. After treatment with AN‐215, tumor volume was 69% (p < 0.01) smaller than that in controls and tumor doubling time was extended from 8.5 ± 0.7 days to 20.3 ± 3.5 days (p < 0.05). Cytotoxic radical AN‐201, carrier RC‐3094 and their unconjugated mixture administered at the same dosage were ineffective. The mortality rate was 12.5% in the AN‐201 group and 16.7% in the group treated with the mixture, but no deaths occurred in mice receiving AN‐215. Because the ester bond linking AN‐201 to the carrier molecule is hydrolyzed much faster in mouse serum than in human serum, in the second experiment we investigated the tolerance to AN‐215 and its effect in nude mice bearing PC‐3 tumors after pharmacological inhibition of serum carboxylesterases. Two applications of AN‐201 at 200 nmol/kg were lethal, whereas no mortality was observed after 4 injections of AN‐215 at the same dose. Administration of 200 nmol/kg AN‐215 on days 1, 7, 17 and 26 again produced 69% tumor inhibition. BN receptors on membranes of PC‐3 tumors were detected by 125I‐[Tyr4]BN binding, and expression of mRNA for BRS‐3 and GRP‐R subtypes was also found. AN‐215 showed a high affinity to PC‐3 tumors, displacing the radioligand at an IC50 of 12.95 ± 0.35 nM. Because BN receptors are present on primary and metastatic prostate cancer, targeted chemotherapy with AN‐215 might benefit patients with advanced prostatic carcinoma who relapsed androgen ablation. Int. J. Cancer 88:652–657, 2000.

Expression of growth hormone-releasing hormone (GHRH) and splice variants of GHRH receptors in human experimental prostate cancers

The expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors in LNCaP, MDA-PCa-2b and PC-3 human prostate cancers grown in nude mice was investigated by RT-PCR. The expression of mRNA for GHRH was detected in LNCaP and PC-3, but not in MDA-PCa-2b prostatic carcinoma. RT-PCR analyses of mRNA isolated from LNCaP, MDA-PCa-2b and PC-3 cancers, revealed the presence of 720 and 566 bp products, corresponding to SV(1) and SV(2) isoforms of GHRH receptors. In PC-3 tumor membranes a radiolabeled GHRH antagonist [125I]-JV-1-42 was bound to one class of high-affinity binding sites (K(d)=1.81+/-0.47 nM) and maximum binding capacity of 332.7+/-27.8 fmol/mg membrane protein. The in vivo uptake of [125I]-JV-1-42 was observed in all xenografts of human prostate cancer, the tracer accumulation being the highest in PC-3 tumors. These results indicate that GHRH and SVs of its receptors, different from those found in the pituitary, are present in experimental human prostate cancers and may form a local mitogenic loop. The antiproliferative effects of GHRH antagonists on growth of prostate cancer could be exerted in part by an interference with this local GHRH system.

Inhibition of Proliferation in Human MNNG/HOS Osteosarcoma and SK-ES-1 Ewing Sarcoma Cell Lines in Vitro and in Vivo by Antagonists of Growth Hormone-Releasing Hormone Effects on Insulin-Like Growth Factor II

Antagonists of growth hormone‐releasing hormone (GH‐RH) can inhibit the proliferation of various tumors either indirectly through the suppression of the pituitary growth hormone/hepatic insulin‐like growth factor I (IGF‐I) axis and the lowering of serum IGF‐I concentration or directly by reducing the levels of IGF‐I and IGF‐II and their mRNA expression in tumors and blocking the effect of autocrine GH‐RH. In this study, the authors investigated the effects of the GH‐RH antagonist JV‐1‐38 on MNNG/HOS human osteosarcoma and SK‐ES‐1 human Ewing sarcoma cell lines.

Expression of mRNA for growth hormone-releasing hormone and splice variants of GHRH receptors in human malignant bone tumors

Splice variants (SV) of receptors for growth hormone-releasing hormone (GHRH) have been found in several human cancer cell lines. GHRH antagonists inhibit growth of various human cancers, including osteosarcomas and Ewings sarcoma, xenografted into nude mice or cultured in vitro and their antiproliferative action could be mediated, in part, through these SV of GHRH receptors. In this study, we found mRNA for the SV(1) isoform of GHRH receptors in human osteosarcoma line MNNG/HOS and SK-ES-1 Ewings sarcoma line. We also detected mRNA for GHRH, which is apparently translated into the GHRH peptide and secreted by the cells, as shown by the presence of GHRH-like immunoreactivity in the conditioned media of cell cultures. In proliferation studies in vitro, the growth of SK-ES-1 and MNNG/HOS cells was dose-dependently inhibited by GHRH antagonist JV-1-38 and an antiserum against human GHRH. Our study indicates the presence of an autocrine stimulatory loop based on GHRH and SV(1) of GHRH receptors in human sarcomas. The direct antiproliferative effects of GHRH antagonists on malignant bone tumors appear to be exerted through the SV(1) of GHRH receptors on tumoral cells.

Inhibition of PC‐3 human prostate cancers by analogs of growth hormone‐releasing hormone (GH‐RH) endowed with vasoactive intestinal peptide (VIP) antagonistic activity

Vasoactive intestinal peptide (VIP) stimulates the proliferation and invasiveness of malignant prostatic cells. Receptors for VIP and the closely related growth hormone‐releasing hormone (GH‐RH) show considerable homology and are found in prostatic and other carcinomas. Among various analogs of GH‐RH synthesized, JV‐1‐52 is a non‐selective VIP/GH‐RH antagonist, whereas JV‐1‐53 is a VIP antagonist devoid of GH‐RH antagonistic effect. In our study, nude mice bearing PC‐3 human androgen‐independent prostate carcinomas were treated with JV‐1‐52 or JV‐1‐53 (20 μg/day, s.c.) for 28 days. Both antagonists produced a similar reduction in tumor volume (62–67%, p < 0.01) and tumor weight (59–62%; p < 0.05) vs. controls and extended tumor doubling‐time from 9.1 to about 16 days (p < 0.05). To investigate the mechanisms involved, in another study we compared the effects of JV‐1‐53 with those of somatostatin analog RC‐160. VIP antagonist JV‐1‐53 reduced tumor weight by 67% (p < 0.01) and suppressed the expression of mRNA for c‐fos and c‐jun oncogenes by about 34% (p < 0.05), without affecting serum levels of insulin‐like growth factor‐I (IGF‐I). In contrast, RC‐160 (50 μg/day) reduced serum IGF‐I by 19% (p < 0.05), but did not significantly decrease tumor weight. mRNA for VIP and high affinity receptors for VIP were detected on PC‐3 tumors. Our results suggest that VIP/GH‐RH antagonists can inhibit the growth of androgen‐independent prostate cancer by abrogating the autocrine/paracrine mitogenic stimuli of VIP. The ability of GH‐RH antagonists to block tumoral VIP receptors, in addition to GH‐RH receptors, could be potentially beneficial for prostate cancer therapy.

Inhibition of in vivo proliferation of MDA-PCa-2b human prostate cancer by a targeted cytotoxic analog of luteinizing hormone-releasing hormone AN-207

The efficacy of therapy with targeted cytotoxic luteinizing hormone-releasing hormone (LHRH) analog AN-207 consisting of superactive doxorubicin derivative AN-201 linked to carrier [D-Lys(6)]LH-RH was evaluated in vivo in nude mice bearing xenografts of MDA-PCa-2b prostate cancer line. AN-207 was administered intravenously (i.v.) at 200 nmol/kg on day 1 and at 150 nmol/kg on day 14. After 4 weeks of treatment with AN-207, tumor growth was inhibited as shown by a 63% (P<0.01) decrease in tumor volume and a 55% (P<0.05) reduction in tumor weight, compared with controls. None of the animals died after administration of AN-207 at the total dose of 350 nmol/kg, and at the end of the experiment the body weights of mice given AN-207 did not differ significantly from controls. A single injection of cytotoxic radical AN-201 at 200 nmol/kg resulted in 43% mortality. In the surviving mice, AN-201 caused a 50% inhibition in tumor volume and a 27% reduction in tumor weight, which were non-significant, as compared to the controls. After 4 weeks, serum prostate-specific antigen concentrations in mice treated with AN-207 were 65% lower than those in controls (P<0.05), while in animals given AN-201 the reduction in serum prostate-specific antigen was only 40% (NS). The expression of mRNA for LHRH receptors was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in MDA-PCa-2b tumors. The present study indicates that chemotherapy targeted to LHRH receptors on tumors inhibits growth of MDA-PCa-2B prostate cancers representative of human carcinoma disseminated to the bone and progressing despite androgen withdrawal.

Effective treatment of experimental U-87MG human glioblastoma in nude mice with a targeted cytotoxic bombesin analogue, AN-215

Some brain tumours, such as glioblastomas express high levels of receptors for bombesin/gastrin releasing peptide. We investigated whether bombesin/gastrin releasing peptide receptors found in glioblastoma cell lines can be utilised for targeting of a cytotoxic bombesin analogue, AN-215 consisting of a potent derivative of doxorubicin, 2-pyrrolino-doxorubicin (AN-201) linked to a bombesin-like peptide carrier. This study reports the effect of AN-215 on the growth of U-87MG human glioblastomas xenografted into nude mice. High affinity binding of AN-215 to U-87MG tumours was characterised by an IC50 value of 4.0±0.1 nM, as determined by radioreceptor assays. mRNA analyses revealed the presence of mRNA for BN receptor subtypes 1 and 2. Treatment with AN-215 significantly (P<0.05) extended tumour doubling time from 4.54±0.2 days to 8.18±1.8 days and inhibited tumour growth as demonstrated by a 69.6% reduction in final tumour volume (P<0.001) and a 64.6% decrease in tumour weight as compared to controls. Cytotoxic radical AN-201 at the same dose was ineffective. The antitumour effect of AN-215 could be blocked by pretreatment with an excess of a bombesin antagonist, indicating that the action of this cytotoxic analogue is receptor-mediated. Our results suggest that patients with inoperable brain tumours such as malignant gliomas may benefit from targeted chemotherapy based on cytotoxic bombesin analogue AN-215.