A cytotoxic analogue of LH-RH, AN-207, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to carrier [D-Lys6]LH-RH, was developed for targeted therapy of cancers expressing LH-RH-receptors. To determine its possible side-effects on the pituitary gland, we investigated the gene expression of pituitary LH-RH-receptors and LH secretion in ovariectomized female and normal male rats after treatment with the maximum tolerated dose of AN-207. The effect of AN-207 on the gene expression of the pituitary GH-RH-receptors and GH secretion was also assessed in male rats. Five hours after a single i.v. injection of AN-207 at 175 nmol/kg, there was a 39-51% decrease in mRNA expression for the pituitary LH-RH-receptors in male and female rats. The carrier, at an equimolar dose, caused a similar reduction (37-39%), whereas the cytotoxic radical AN-201, at an equitoxic dose (110 nmol/kg), produced only a 12-24% decrease (NS) in the mRNA expression of LH-RH-receptors. AN-207 and the carrier analogue induced a comparable 90-100-fold increase in serum LH concentrations in male rats, and the same 12-fold elevation in OVX rats at 5 h. Seven days after treatment with AN-207, the mRNA levels for the LH-RH receptors and the serum LH concentration were back to normal in both sexes. AN-207, the carrier, and AN-201 had no significant effect on the expression of mRNA for GH-RH-receptors in the pituitary. In vitro, a continuous perfusion of pituitary cells with 10 nM AN-207 did not affect the hormone-releasing function of the targeted LH cells or the nontargeted GH cells. Our results demonstrate that cytotoxic LH-RH analogue AN-207, at the maximum tolerated dose causes only a transient decrease in the gene expression of the pituitary LH-RH receptors, and the levels of mRNA for LH-RH receptor fully recover within 7 days. Moreover, the carrier hormone moiety, and not the cytotoxic radical in AN-207 is responsible for this transient suppression. Our findings suggest that the therapy with cytotoxic LH-RH analogues will not inflict permanent damage to pituitary function.

PURPOSE:The expression of somatostatin receptors (SSTRs) allows the localization and treatment of some tumors with radiolabeled SST analogues. We investigated whether SSTRs on human pancreatic cancer lines xenografted into nude mice can be used for targeting of cytotoxic somatostatin analogue AN-238, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to octapeptide carrier RC-121.EXPERIMENTAL DESIGN:AN-238 and AN-201 were administered i.v. to nude mice bearing SW-1990 pancreatic cancers. Tumor growth reduction and survival were analyzed, and cell proliferation and apoptosis were determined with histological methods. The effects of repeated administration of AN-238 and AN-201 were also evaluated on xenografted Panc-1, MiaPaCa-2, CFPAC-1, Capan-1, and Capan-2 pancreatic cancers. The expression of mRNA for SSTR subtypes 2A, 3, and 5 in tumors was analyzed by reverse transcription-PCR, and binding assays were performed.RESULTS:All of the cancer models except MiaPaCa-2 displayed functional receptors for SST. SW-1990 expressed mRNA for SSTR subtypes 3 and 5, whereas various patterns of subtypes 2A, 3, and 5 were found in other pancreatic cancers. Repeated administration of AN-238 at 150 nmol/kg significantly inhibited growth of SW-1990 cancers (93% after 45 days; P = 0.016) and other tumors but not MiaPaCa-2. AN-201 was toxic and less effective. The efficacy of AN-238 was consistent with SSTR expression.CONCLUSIONS:Growth of experimental human pancreatic cancers that express SSTRs can be inhibited by cytotoxic somatostatin analogue AN-238.

Cytotoxic analogs of somatostatin (SST), such as AN‐238, which consists of 2‐pyrrolinodoxorubicin (AN‐201) linked to the SST carrier RC‐121, can be targeted to tumors that express SST receptors. Because SST receptors are present in ovarian carcinoma cells, the authors evaluated the effect of AN‐238 on the UCI‐107 ovarian carcinoma cell line.

We evaluated the effects of GHRH antagonists on the proliferation of MiaPaCa-2 human pancreatic cancer cells and cAMP signaling in vitro. GHRH antagonists inhibited the proliferation of MiaPaCa-2 cells in vitro in a dose-dependent way and caused a significant elevation in cAMP production. In a superfusion system, short-term exposure of the cells to GHRH antagonists evoked an acute, dose-dependent release of cAMP into the medium. Native GHRH, which stimulates cAMP efflux from pituitary at nanomolar doses, did not influence cAMP release from cultured or superfused MiaPaCa-2 cells even at 10–30 μM. VIP, PACAP, secretin and glucagon also did not influence cell proliferation or cAMP production. Adenylate cyclase activator forskolin (FSK) caused a greater cAMP response, but a smaller antiproliferative effect than GHRH antagonists. Combined treatment with FSK and GHRH antagonist JV-1-38 potentiated the cAMP-inducing effect of FSK, but did not produce a greater inhibition of cell proliferation than JV-1-38 alone. A selective accumulation of radiolabeled GHRH antagonist [125I]JV-1-42 in vivo in MiaPaCa-2 carcinoma xenografted into nude mice was also observed. In conclusion, second messengers other than cAMP participate in the signal transduction pathways of GHRH analogs mediated by tumoral GHRH receptors.

BACKGROUND:Prostate-specific antigen (PSA) is the best tumor marker for diagnosis and prognosis of prostatic carcinoma. The secretion of PSA from LNCaP human prostate cancer cells is influenced by acute stimuli such as vasoactive intestinal peptide (VIP), growth hormone-releasing hormone (GHRH), and chronic stimuli like androgens.METHODS:To study the regulation of basal and VIP/GHRH or androgen-stimulated secretion from LNCaP cells, we used a superfusion system, which allowed us to simultaneously measure PSA gene expression, PSA secretion, and cAMP release from the same cancer cells. LNCaP cancer cells were also implanted orthotopically into nude mice.RESULTS:VIP (30 pM-3 nM), GHRH (3 nM-300 nM), and dihydrotestosterone (100 nM) induced a significant increase in PSA gene expression, PSA secretion, and cAMP release. The dose and time-dependent effects of peptides were manifested only in the presence of androgens. At the end of continuous stimulation of cells with 1 nM VIP for 2 hr, large amounts of stored immunoreactive PSA still remained in the cells. Adenylate cyclase activator, forskolin (FSK), significantly increased PSA secretion and gene expression, and potassium, which causes nonspecific depolarization of membranes, augmented gene expression, and secretion of PSA, but did not influence cAMP release. This suggests that PSA secretion is regulated by cAMP-dependent as well as cAMP-independent pathways. In superfusion system, stimulatory effects of VIP and GHRH on PSA secretion were inhibited by VIP antagonist JV-1-53, and less by GHRH antagonist JV-1-38. In cell cultures, JV-1-38 had a stronger inhibitory effect on proliferation, indicating an involvement of the recently discovered tumoral GHRH receptors in this process. In nude mice, with orthotopically implanted LNCaP cancer cells, GHRH antagonist JV-1-38 alone or androgen ablation by castration had no effect on tumor growth and PSA levels. However, castration combined with treatment with GHRH antagonist, significantly decreased tumor growth and PSA secretion.CONCLUSIONS:Our findings suggest that the secretion of PSA is regulated rather than constitutive, contrary to previous reports. In addition, the effect of GHRH and VIP antagonists on PSA secretion from prostate cancer cells is not correlated with their antiproliferative action.

The mechanisms through which LH-RH antagonists suppress gonadotroph functions and LH-RH receptor (LH-RH-R) production are incompletely understood. To elucidate these mechanisms, we investigated the effects of Cetrorelix on the mRNA expression of pituitary LH-RH-R and luteinizing hormone (LH) secretion in three experimental systems with different pituitary LH-RH environments. Ovariectomy induced 3.61-fold and 6.34-fold increases in the mRNA expression of pituitary LH-RH-R in rats after 11 and 21 days, respectively. After (5 h) a single injection of 100 microg Cetrorelix, no significant decrease occurred in the mRNA levels of pituitary LH-RH-R in ovariectomized (OVX) rats with high pituitary exposure to LH-RH, but there was a significant 23.2% reduction in cycling rats with normal hypophysial LH-RH environment. Prolonged treatment for 10 days with a Cetrorelix depot formulation releasing 100 microg/day decreased the concentration of mRNA for pituitary LH-RH-R by 72.6% in OVX rats, but only by 32.9% in normal rats. The decline in serum LH was 98.7% in OVX rats and 63.2% in normal rats, resulting in a minimal 0.1--0.2 ng/ml LH concentration in both groups. A continuous exposure of pituitary cells to 100 nM Cetrorelix in the superfusion system, which is devoid of LH-RH, did not cause any significant changes in LH-RH-R mRNA level. These studies demonstrate that prolonged exposure to Cetrorelix in vivo, but not in vitro, down-regulates the mRNA expression of the pituitary receptors for LH-RH. Our findings indicate that LH-RH antagonists exert their inhibitory effects on the gene expression of pituitary LH-RH-R by counteracting the stimulatory effect of endogenous LH-RH.