Gordan Srkalovic

Inhibitory effects of analogs of luteinizing hormone-releasing hormone and somatostatin on pancreatic cancers in hamsters events that accompany tumor regression

Syrian golden hamsters bearing N‐nitrosobis(2‐oxopropyl)amine (BOP)‐induced pancreatic carcinomas were treated for 2 months with the delayed delivery systems of the agonist D‐Trp‐6‐LH‐RH (microcapsules releasing 25 μg/day for 30 days), the somatostatin analog RC‐160 (the microcapsules liberating 48.2 μg/day for 30 days), or with the combination of these two analogues. The increase in the dose of RC‐160 was possible in view of the lack of toxicity of this analog. This higher dose of RC‐160 exerted a greater suppressive effect on pancreatic cancers than the regimens previously used (5‐25 μg/day). RC‐160, D‐Trp‐6‐LH‐RH, and their combination reduced the number of pancreatic carcinomas and significantly inhibited tumor growth as compared with the controls. The combination had the strongest tumor‐inhibitory effect and reduced tumor weight by 85% as compared with controls. Both the light and electron microscopic analysis of the tumors showed that the inhibitory effect was due to the enhancement of apoptosis (programmed cell death) of tumor cells. Insulin‐like growth factor (IGF‐I) receptors were detected immunohistochemically in the untreated tumors and their number decreased after the treatment with the analogues. Binding of D‐Trp‐6‐LH‐RH and RC‐160 to tumor cells was shown immunohistochemically and receptors to these analogues and IGF‐I were also determined biochemically by radioligand titration. Treatment with D‐Trp‐6‐LH‐RH and RC‐160 decreased the binding capacity of receptors for D‐Trp‐6‐LH‐RH and IGF‐I, producing down‐regulation of these receptors. This suggests that pancreatic tumor cells with receptors to these peptides are sensitive to the treatment. This work reinforces the view that the combination of high doses of somatostatin analog RC‐160 with LH‐RH agonists or antagonists should be considered for the development of a new hormonal therapy for ductal pancreatic cancers.

Antitumor effects of analogs of LH-RH and somatostatin: Experimental and clinical studies

Many clinical approaches for the treatment of hormone-sensitive tumors are being developed based on analogs of LH-RH and somatostatin. Inhibition of the pituitary-gonadal axis forms the basis for oncological applications of LH-RH agonists like [D-Trp6]-LH-RH and new LH-RH antagonists free of edematogenic effects such as [Ac-D-Nal(2)1-D-Phe(4Cl)2-D-Pal(3)3,D-Cit6,D-Ala10]-LH -RH (SB-75). Agonists and antagonists of LH-RH have been used in patients with prostate cancer and might be also beneficial for the treatment of breast cancer and ovarian, endometrial and pancreatic carcinomas. Some of the effects of LH-RH analogs can be due to direct action since LH-RH receptors have been found in these cancers. The use of sustained delivery systems based on microcapsules of PLG, makes the treatment more efficacious. Octapeptide analogs of somatostatin such as D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160) and related analogs were designed specifically for antitumor activity. These somatostatin analogs, by virtue of having a wide spectrum of activities appear to inhibit various tumors through multiple mechanisms. Direct antiproliferative actions of somatostatin analogs appear to be mediated by specific receptors located on tumor cells. High affinity binding sites for RC-160 and related analogs have been found in human pancreatic, prostate, breast and ovarian cancers and brain tumors such as meningiomas. In vivo administration of analog RC-160 inhibits the growth of Dunning R-3327 prostate cancers in rats, MXT mammary tumors in mice and BOP-induced ductal pancreatic cancers in hamsters. Combination of microcapsules of RC-160 with [D-Trp6]-LH-RH results in synergistic potentiation of the inhibition of these cancers. Somatostatin analog RC-160 and LH-RH antagonist SB-75 are the object of further experimental studies and clinical trials aimed at the exploration of their inhibitory effects on the processes of malignant growth.

Recovery of Pituitary-Gonadal Function in Male and Female Rats after Prolonged Administration of a Potent Antagonist of Luteinizing Hormone-Releasing Hormone (SB-75)

The reversibility of the antifertility effects induced by long-term administration of the LH-RH antagonistic analog [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]-LH-RH (SB-75) was investigated in male and female rats. Male rats were implanted with osmotic minipumps releasing 50 micrograms of SB-75/day for 60 days. The control rats were implanted with minipumps containing only vehicle. The treatment with the antagonist caused a significant decrease in the weights of the testes, seminal vesicles and ventral prostates (p less than 0.01) and reduced serum LH and testosterone levels (p less than 0.01). The histology of the testes from the treated rats showed that spermatogenesis was totally depressed. No mature elongated or round spermatids were found in the seminiferous tubules, spermatocytes being the most advanced germ cell form in 100% of the testicular tubules. These changes indicate that a total spermatogenetic arrest occurred in the treated animals. Ninety days after cessation of treatment with the LH-RH antagonist, there was a complete recovery of the weights of the testes, seminal vesicles and ventral prostates and LH and testosterone returned to control levels. Histological studies revealed a complete recovery of spermatogenesis, with 99.2% of seminiferous tubules containing mature elongated spermatids. Immediately after the discontinuation of treatment with SB-75, a significant down-regulation of the pituitary LH-RH receptors was found, but 90 days later, this phenomenon was completely reversed. Female rats were injected every 3 weeks for 6 weeks with SB-75 microcapsules, at a dose calculated to release 27 micrograms/day of the antagonist. The treatment with SB-75 disrupted the normal estrous cycle. Body weights were not affected, but ovarian and uterine weights were significantly decreased (p less than 0.01 and p less than 0.05, respectively) in the animals treated with the antagonist. Treated rats had significantly lower LH (p less than 0.05) and estradiol (p less than 0.01) levels than controls. The histology of the ovaries from the SB-75-treated group showed that the ratio of small to large maturing follicles increased significantly (p less than 0.01) and corpora lutea were absent. Two months after the cessation of treatment, a complete recovery in the organ weights and in hormonal levels was observed and no histological differences were found between the ovaries in treated and untreated rats. These collective results indicate that the suppression of gonadal function induced by the treatment with LH-RH antagonist SB-75 is completely reversible both in male and female animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Receptors for D-Trp6-luteinizing hormone-releasing hormone, somatostatin, and insulin-like growth factor I in MXT mouse mammary carcinoma.

Abstract Membrane receptors for d-Trp6-luteinizing hormone-releasing hormone (d-Trp6-LH-RH), somatostatin-14 (SS-14), and insulin-like growth factor I (IGF-I) were estimated in MXT mammary cancers of mice using sensitive multipoint micromethods. The receptors were characterized in untreated animals and following in vivo treatment with microcapsules of the agonist d-Trp6-LH-RH and the somatostatin analog RC-160, which strongly inhibited tumor growth. In the control group, d-Trp6-LH-RH was bound to the single class of saturable, specific, noncooperative receptor sites (Kd , = 29.3 ± 8.48 × 10-9 M; Bmax = 4.55 ± 0.31 pmol/mg membrane protein). Treatment with d-Trp6-LH-RH alone or in combination with RC-160 produced down-regulation of membrane receptors for d-Trp6-LH-RH on MXT mammary tumor cells. RC-160 alone and ovariectomy were without effect on d-Trp6-LH-RH receptors. On the membrane surface of MXT mammary cells, we found one class of high affinity, specific, saturable binding sites for SS-14 (Kd = 4.4 ± 1.9 × 10-9 M; Bmax = 0.58 ± 0.21 pmol/mg membrane protein). Treatment with RC-160 alone or combined with d-Trp6-LH-RH significantly increased both the dissociation binding constant (K d = 18.6 ± 3.5 × 10-9 and 10.1 ± 0.7 × 10-9 M, respectively) and the binding capacity (Bmax = 13.98 ± 1.7 and 21.00 ± 4.0 pmol/mg membrane protein, respectively). We also found specific binding sites (K d = 3.01 ± 0.15 × 10-9 M; Bmax = 2.24 ± 0.96 pmol/mg membrane protein) for IGF-I in the membrane fractions of MXT mammary cancers. Chronic treatment with d-Trp6-LH-RH and RC-160 alone or in combination, as well as ovariectomy, significantly decreased the dissociation binding constant of IGF-I membrane receptors on MXT mammary cells. Our results strongly suggest an important role of LH-RH, SS-14, and IGF-I in the growth of MXT mammary carcinoma. Changes in characteristics of receptors after treatment with analogs of LH-RH and SS-14 along with tumor growth inhibition provide additional support for the direct effect of these peptides on tumor cells. A possible significance of these findings as applied to a clinical environment is discussed.