Growth hormone-releasing hormone antagonists inhibit growth of human ovarian cancer.

Epithelial ovarian carcinoma is the leading cause of cancer-related deaths among women with gynecologic malignancies. Antagonists of the growth hormone-releasing hormone (GHRH) have been shown to inhibit growth of various cancers through endocrine, autocrine, and paracrine mechanisms. In this study, we have investigated the effects of GHRH antagonists (GHRHa) in ES-2 human clear cell ovarian cancer and in UCI-107 human serous ovarian cancer in vitro and in vivo. We evaluated the expression of mRNA for GHRH receptor, the binding to GHRH receptors, in specimens of ES-2 ovarian cancer. We evaluated also the in vitro effects of GHRHa on ES-2 cells and the in vivo effect of 2 different GHRHa on ES-2 and UCI-107 tumors. Nude mice bearing xenografts on ES-2 and UCI-107 ovarian cancer were treated with JMR-132 and MZ-J-7-118, respectively. Tumor growth was compared to control. ES-2 cells expressed mRNA for the functional splice variant SV1 of the GHRH receptor. JMR-132 inhibited cell proliferation in vitro by 42% and 18% at 10 and 1 µM concentration, respectively. Specific high affinity receptors for GHRH were detected in ES-2 cancer samples. In vivo daily subcutaneous injections of GHRHa significantly reduced tumor growth compared to a control group in both animal models. Our results indicate that GHRHa such as JMR-132 and MZ-J-7-118 can inhibit the growth of human ovarian cancer. The efficacy of GHRHa in ovarian cancer should be assessed in clinical trials.

The inhibitory effect of a novel cytotoxic somatostatin analogue AN-162 on experimental glioblastoma.

Glioblastoma multiforme is the most common and most aggressive type of high grade tumor with a poor prognosis upon discovery. Based on earlier promising results earned with AN-162, a doxorubicin molecule linked to somatostatin (SST) analogue RC-160, it was our aim to determine the effect of AN-162 on DBTRG-05 glioblastoma cell line, and to test its efficacy in experimental brain tumors. We detected the expression of mRNA for somatostatin receptor (SSTR) subtypes 2 and 3 in DBTRG-05 cells with RT-PCR. Using ligand competition assay, specific high affinity receptors for somatostatin were found. The MTT assay showed that both AN-162 and doxorubicin (DOX) significantly inhibited cell proliferation and that there was no significant difference between the effects in vitro. Nude mice were xenografted with DBTRG-05 glioblastoma tumors. AN-162 showed a significant inhibition of tumor growth compared with the control group and the groups treated with equimolar doses of doxorubicin, somatostatin analogue RC-160, or the unconjugated mixture of doxorubicin plus RC-160. The tumor doubling time in the group of animals treated with AN-162 was extended and was significantly different from doubling times in the control group and in the other treatment groups. Our study clearly demonstrates a potent inhibitory effect of AN-162 in experimental glioblastoma, thus suggesting the possibility of its utilization in patients suffering from malignant brain cancer.

Extremely high maternal alkaline phosphatase serum concentration with syncytiotrophoblastic origin.

An extremely high alkaline phosphatase (AP) concentration (3609 IU/litre) was found in a 20 year old primigravida at 37 week's gestation, prompting an examination of its histological and cellular origin. Immunohistochemistry and western blots using antibodies against AP, Ki-67, phospho-protein kinase B (Akt), phospho-p44/42 mitogen activated protein kinase/extracellular signal regulated kinase 1/2 (MAPK/Erk1/2), phospho-glycogen synthase kinase-3beta (GSK-3beta), phospho-stress activated protein kinase/c-Jun N-terminal kinase, total-Akt, total-GSK-3beta, and phospho-p38-MAPK were carried out on index and control placental samples of the same gestational age. Compared with controls, staining of the index placenta showed minimal AP labelling of the brush border and remarkable positivity of the intervillous space. Cytotrophoblastic proliferation was 8-10% in the index placenta compared with 1-2% in controls. The index placenta also had raised concentrations of protein kinases with important roles in cell differentiation. The proliferation and differentiation rates of the cytotrophoblasts were found to be five times higher in index samples than in controls. It is hypothesised that loss of syncytial membranes in immature villi led to increased AP concentrations in the maternal circulation and decreased AP staining of the placenta. Loss of the syncytium might also stimulate increased proliferation of villous cytotrophoblasts, which would then fuse and maintain the syncytium.

Overexpression of placental tissue protein 17b/TIP47 in cervical dysplasias and cervical carcinoma.

In our previous Western- and Northern-blot investigations, high over-expression of placental protein 17b/TIP47 was detected in extracts of human cervical carcinoma tissues compared to normal conditions of the cervical tissue. PP17b serum levels were also elevated in untreated cervical carcinoma patients compared to healthy controls. In the present study, the expression pattern of PP17 proteins was investigated in various cervical dysplasias and in cervical carcinoma tissue specimens by the streptavidin-biotin immunoperoxidase technique using PP17-specific antiserum. In normal third-trimester human placentas, which served as positive controls, mainly cytoplasmic PP17 immunostaining of syncytiotrophoblasts and chorionic trophoblasts was observed. Normal human uterine cervical squamous and glandular epithelia were negative or weakly positive, while in low grade dysplasias (CIN I-II) only the cytoplasms of dysplastic cells were weakly positive or positive; in high grade dysplasias (CIN III/ISC) cytoplasms of the dysplastic cells were strongly positive. Normal and superficial cells in the differentiated zones were negative in all tissue specimens. In cases of invasive epithelial cervical carcinomas, small basal-type tumour cells were mostly negative whilst cells with squamous differentiation were strongly positive for PP17. Our hypotheses for this newly detected phenomenon are briefly discussed.

Molecular cloning and characterization of placental tissue protein 18 (PP18a)/human mitochondrial branched-chain aminotransferase (BCATm) and its novel alternatively spliced PP18b variant.

Five different insert-length cDNAs encoding for soluble placental tissue protein 18 (PP18) variants were isolated by screening a human placental cDNA library using monospecific anti-PP18 serum. Sequence analysis of the longest clone showed that the insert contains an open reading frame encoding for a 392 residue-long protein with a 27 amino acid mitochondrial targeting sequence. The mature protein-designated PP18a-is 41.264 kDa consisting of 365 residues and is identical to the previously isolated and characterized PP18 antigen described in 1985. We also found a new, alternatively spliced cDNA encoding for a 300 residue-long, 33.776 kDa protein, which was designated PP18b. Alignment search of the protein databank showed that PP18a is almost entirely identical to the human mitochondrial branched-chain aminotransferase, while PP18b is its newly discovered splicing variant. We detected the two PP18 variants in normal adult and fetal human tissues besides the mitochondrial (only PP18a) and cytosolic (only PP18b) fractions of term placenta with chemiluminescence Western blot analysis. The 41 kDa PP18a variant was expressed ubiquitously, while the 33 kDa PP18b variant was found in smaller amounts in nearly all tissues. Trace amounts of the variants were present in the sera of non-pregnant healthy controls, as well as in pregnant women, but there was no real change in serum levels during pregnancy. In conclusion, PP18 variants are not specific for the placenta. Aminotransferase activity of placental origin PP18 antigens was verified by structural analysis and by a coupled branched-chain aminotransferase/glutamate dehydrogenase assay.

Hormonal and clinical effects of chronic gonadotropin-releasing hormone agonist treatment in polycystic ovary syndrome.

The aim of the study was to evaluate the hormonal (focusing on the urinary steroid profile) and clinical effects of chronic gonadotropin-releasing hormone (GnRH) agonist treatment in patients with polycystic ovary syndrome (PCOS) suffering from hirsutism. A long-acting GnRH agonist was administered for 6 months in eight PCOS patients. Hormonal effects were measured by determining serum luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, testosterone and estradiol concentrations, and by profiling urinary steroids using capillary gas chromatography of 24-hour urine samples. To evaluate 5 alpha-reductase enzyme activity, the ratios of androsterone to etiocholanolone and 5 alpha-tetrahydrocortisol to tetrahydrocortisol were calculated in urine samples. The ratio of androgen to cortisol metabolites was also determined before, and 3 and 6 months after therapy. LH and estradiol levels were suppressed significantly after the first injection and testosterone after the second injection of the GnRH agonist. Thus, serum testosterone was normalized. Ratios of urinary steroids reflecting 5 alpha-reductase enzyme activity (androsterone to etiocholanolone and 5 alpha-tetrahydrocortisol to tetrahydrocortisol) and the ratio of androgen to cortisol metabolites decreased significantly after 3 months of treatment. Degree of hirsutism, assessed by Ferriman-Gallwey score, diminished after 6 months, but not significantly. In conclusion, our data show that long-acting GnRH agonist treatment of PCOS patients is effective in reducing serum and urinary androgen levels, but it is not accompanied by an effective reduction in hirsutism during a 6-month treatment period. A longer or a combined treatment would be needed to achieve significant improvement in hirsutism. Gas chromatographic profiling of urinary steroids and the use of specific ratios of the excreted metabolites seems to be a sensitive tool both in the diagnosis of PCOS and in monitoring ovarian suppression.