Pancreatic cancer cells require an EGF receptor-mediated autocrine pathway for proliferation in serum-free conditions

In-vitro and in-vivo studies have shown that autocrine growth factors and receptors are frequently expressed in human malignancies. Few of these studies, however, provide evidence that the identified autocrine pathway is functional. In this study, a functional autocrine growth pathway in pancreatic cancer has been identified using an in-vitro cell culture system. When pancreatic cancer cells were grown without change of medium, proliferation was greater than when either medium was replaced frequently (HPAF, CAPAN-2, PANC-1 or SW1990) or cells were grown in the presence of the EGF receptor tyrosine kinase inhibitor AG1478 or the MEK inhibitor PD098059 (HPAF or CAPAN-2). Activity of extracellular-regulated kinases (ERK) 1 and 2 and c- jun and c- fos mRNA levels were significantly elevated in CAPAN-2 cells cultured continuously in serum-free medium. Collectively, the observations indicate that the EGF receptor and the ERK MAP kinase pathway mediate autocrine signals. In contrast to previous reports, the GRP and IGF-I receptors were shown not to be required for autocrine effects on pancreatic cancer cell proliferation. Autocrine stimulation of the EGF receptor can contribute to sustained mitogenic activity and proliferation of pancreatic cancer cells.     

Targeting of doxorubicin to ES-2 human ovarian cancers in nude mice by linking to an analog of luteinizing hormone-releasing hormone improves its effectiveness

Receptors for luteinizing hormone-releasing hormone (LHRH), expressed by ovarian cancers, can be used for targeting chemotherapeutic compounds more selectively to these tumors. We investigated the effects of cytotoxic LHRH analog AN-152, consisting of doxorubicin (DOX)-14-O-hemiglutarate linked to the epsilon-amino group of [D-Lys6]LHRH, on the growth of LHRH receptor-positive ES-2 human ovarian cancer line xenografted into nude mice. A single injection of AN-152, at a dose of 345 nmol/20 g body weight, caused a 34.5% reduction (P<0.05) in tumor growth after 28 days, while its cytotoxic moiety DOX was inactive at the same dose. Since the overexpression of certain growth factors and/or their receptors, such as vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and HER-2/neu, as well as various oncogenes like c-fos and c-jun, is associated with unfavorable prognosis and contributes to progressive growth of ovarian carcinomas, their mRNA levels were analyzed by RT-PCR. Treatment with AN-152 significantly (P<0.05) reduced the expression of EGFR, VEGF, c-fos and c-jun, to 49%, 48%, 55% and 58% respectively, compared to controls. HER-2/neu mRNA expression was also decreased to non-detectable levels. Conversely, DOX decreased non-significantly the expression levels for EGFR by 32%, VEGF 35%, both c-fos and c-jun approximately 20% and HER-2/neu by only 15%. In conclusion, cytotoxic LHRH analog AN-152 could be considered for chemotherapy of ovarian cancers expressing LHRH receptors.     

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.     

Inhibition of growth of human malignant glioblastoma in nude mice by antagonists of bombesin/gastrin-releasing peptide

The effects of antagonists of bombesin/gastrin-releasing peptide (GRP) on the growth of human malignant glioblastoma cell line U-87MG xenografted into nude mice were evaluated. Nude mice bearing s.c. implanted U-87MG tumors were treated with bombesin/GRP antagonists RC-3095 and RC-3940-II. RC-3095 and RC-3940-II administered s.c. at a dose of 20 micrograms/day for 4 weeks decreased the volume of U-87MG xenografts by 60 and 74%, respectively, compared with controls. RT-PCR analysis showed that U-87MG xenografts expressed mRNA for bombesin receptor subtype (BRS)-1 (GRP receptor) and BRS-2 (neuromedin-B receptor), but the mRNA for GRP ligand was not detected in U-87MG cells suggesting that GRP may stimulate the growth of U-87MG glioblastomas by a paracrine mechanism. The levels of mRNA for c-fos oncogene were decreased by 30-40% in U-87MG tumors treated with RC-3095 or RC-3940-II. In U-373MG glioblastoma cells, which also express BRS-1, and U-87MG cells, cultured in vitro, GRP(14-27) induced the expression of c-fos mRNA, and some c-jun mRNA, in a time-dependent manner with the maximal effect occurring 2 h after the stimulation and a return to basal levels after 8 h. Antagonist RC-3940-II inhibited the stimulation of c-fos by GRP(14-27). Our results indicate that antagonists of bombesin/GRP inhibit the growth of U-87MG glioblastomas by a mechanism that may involve the downregulation of c-fos oncogene.     

Correlation between leuteinizing hormone responses to luteinizing hormone-releasing hormone (LH-RH) and to D-leu-6-LH-RH-ethylamide in patients with hypothalamo-pituitary disease.

Seventeen patients (eleven females and six males) with organic hypothalamo-pituitary disease were subjected to a test consisting of a rapid intravenous injection of 50 mug of luteinizing hormone-releasing hormone (LH-RH) at 8:00 A.M., with blood sampling before and 30 and 60 minutes afterward. Two to four weeks later, a rapid intravenous injection of 50 mug of D-Leu-6-des-Gly-10-LH-RH-ethylamide (D-Leu-6-LH-RH-EA) was given under similar conditions, with blood sampling before and 30, 60, and 90 minutes afterward. Serum LH levels were determined by radioimmunoassay. D-Leu-6-LH-RH-EA caused a greater and more sustained rise in serum LH levels than did an equal dose of LH-RH. However, functional classifications of patients were similar with either preparation. This finding suggests that acute administration of D-Leu-6-LH-RH-EA does not cause a higher number of relevant responses as compared with LH-RH, but only a greater stimulation of LH releases in responsive patients.     

Attempts to induce ovulation in amenorrheic patients using D-Ala-6-LH-RH propylamide.

Thirteen amenorrheic patients, two with primary amenorrhea, and eleven with secondary amenorrhea, including five with anorexia nervosa, were treated with an analog of LH-RH, D-Ala-6-desGly-10-LH-RH propylamide (D-Ala-6-LH-RH PA). The patients were follwed with checks of daily basal body temperature, cervical mucus characteristics, urinary total estrogens and pregnanediol, plasma LH and FSH, and twice-weekly clinical checkups. D-Ala-6-LH-RH PA was given continuously for 11 days at a dose of 50 microgram im from the 4th day of a steroid-induced cycle. Ethynyl estradiol (50 microgram orally) was given twice on day 14 followed by 250 microgram of D-Ala-6-LH-RH PA for the following 3 days. All the treated cycles were monophasic. Withdrawal bleeding occured in nine patients from the 4th to 6th days posttreatment. A significant total maximal increment in estrogens was found in seven patients, which was higher than the values obtained previously in the same patients with human menopausal gonadotropin. One patient conceived in the cycle immediately following discontinuation of treatment, indicating a possible effect of analog on the follicular development during the treatment; a normal baby was delivered. A second patient conceived three cycles afterwards, and two other patients began normal menstrual cycles.     

Levels of luteinizing hormone (LH), follicle-stimulating hormone, and 17 beta-estradiol in response to D-Trp6-LH-releasing hormone during different phases of the menstrual cycle in normal women.

The response of serum LH, FSH, and 17beta-estradiol (17b-E) to stimulation with D-Tryp6-LH-releasing hormone (LH-RH analog) was measured in 34 normal women. The analog was administered either by vein, muscle, or continuous iv infusion in doses from 1 to 50 mcg during different phases of the menstrual cycle. The increase in LH and FSH to 10 mcg analog was significant (p .05) in all phases of the menstrual cycle by 60 minutes postadministration and lasted for at least 8 hours. 17b-E increased significantly by 8 hours. LH-RH analog was 40 and 21 times more potent than LH-RH in the stimulation of LH and FSH, respectively, during the follicular phase. In the ovulatory phase, LH and FSH responses differed kinetically from their responses in the other phases, and gonadotropin release was both faster and greater. The gonadotropin responses of 3 men when compared with those of the female subjects were almost always smaller (p .05). Because of the greater potency of this LH-RH analog, it may be useful in the treatment of infertility.