The somatostatin receptor subtype 2 is expressed in normal and tumoral human tissues70.

Somatostatin (SS) can inhibit growth hormone (GH) secretion from the pituitary and tumor cell proliferation via membrane-bound receptors (SST). Five SST subtypes have been cloned and can be discriminated by specific peptides. In order to evaluate the human tissue distribution of the SSTs, we first used the cross-linking assay with the 125I-SS-14. A cross-linked complex of 57 kDa was detected in a majority (76%) of the surgical biopsies of normal and tumoral tissues examined (N = 222) and in all tested cell lines (N = 20). However, in regard to the organs, the incidence varied from 33% (epiploon metastases) to 100% (colorectal adenocarcinoma, prostate). Additional, minor SS-14 cross-linked complexes were detected in a few samples, suggesting the simultaneous existence of other SST subtypes. In tumor cell lines, the 57-kDa complex was reduced by the SST2-selective SS analogs BIM23014, BIM23060, and BIM23068, and by SS-14 but not by the non-SST2-selective BIM23052 and BIM23056. Its pharmacological profile therefore corresponded to SST2. Northern blot analysis showed one 2.5-kb human SST2 mRNA in these cell lines. We demonstrate that SST2 is detectable in normal and tumoral human tissues and thus represents an SST subtype target for the development of more specific SS analogs.

Somatostatin-14 mainly binds the somatostatin receptor subtype 2 in human neuroblastoma tumors.

Neuroblastoma is a pediatric cancer for which a cure is elusive for most children with disseminated disease. Neuroblastomas possess receptors for somatostatin (SS). Some SS analogues can inhibit their proliferation. In addition, when SS analogues were used as agents for scintigraphy, neuroblastoma tumor sites can be localized with high efficiency. In this study, to better characterize the SS receptor subtype(s) (sst1-5) present in primary tumors and metastases of neuroblastoma, we show that: (1) The ligand 125I-Tyr11-SS-14 binding on membrane proteins from primary tumors and metastases of neuroblastoma cell line IGR-N-91 developed in nude mice shows similar values of Kd (in order of 0.1 nM) and Bmax (in order of fmol/mg) by filter-retention assay. These data are close to those measured on two other neuroblastoma cell lines: SK-N-SH and IGR-N-835 or to that measured on the rat cerebral cortex. (2) The IGR-N-91 sublines derived from primary tumor and metastases show one major complex of 57 kD by the chemical cross-linking assay using the ligands: 125I-SS-14 and 125I-BIM23014. One similar major complex of 57 kD was also detected in SK-N-SH and IGR-N-835 or in the cerebral cortex. (3) Addition of excess nonlabeled peptides selective for sst2 (BIM23014, BIM23060, BIM23068) suppressed the formation of the complex 57 kD whereas addition of BIM23052 or BIM23056 (sst5 and sst3 selective respectively) does not. This pharmacological profile corresponds to sst2. (4) Only RNA message of sst2 gene is detected in IGR-N-91 cells and its metastases derived sublines by reverse-transcription-polymerase chain reaction and Northern hybridization in keeping with the presence of sst2. (5) In human biopsies, the complex of 57 kD corresponding to sst2 is consistently detected in three samples of the histological subset of the disease: benign ganglioneuroma, ganglioneuroblastoma and immature neuroblastoma. Therefore, the sst2 should be considered as the primary target to develop more potent SS analogues for neuroblastoma therapy or/and scintigraphy.

Alteration of prostaglandin E receptors in advanced breast tumour cell lines.

We studied the direct effects of PGE2, often produced at high levels by mammary tumours, on three human breast cancer cell lines diversely advanced in malignancy regarding differentiation and tumorigenicity in nude mice. We evaluated PGE2 effect on cell growth, PGE2 receptor level and functionality. Our results show that PGE2 induces cAMP accumulation and inhibits the growth of the most differentiated breast cancer cells. We also demonstrate that loss and probably dysfunction of PGE2 receptors is related to an advanced tumorigenic phenotype of the cells. Thus, it seems that during progression of breast cancer, the cell growth escapes from control by PGE2. Nevertheless, it is possible to control the growth of advanced breast cancer cells in vitro by direct induction of intracellular cAMP accumulation.

Evidence for separate mechanisms of antiproliferative action of indomethacin and prostaglandin on MCF-7 breast cancer cells.

Indomethacin is reported to decrease the growth of many tumour cell lines. It is also known to be an anti-inflammatory agent acting by the inhibition of prostaglandin (PG) synthesis. To evaluate a clinical application as antitumoral agent, we have studied whether antiproliferative effect of indomethacin in breast cancer is related to its action on the prostaglandin production. We have observed that indomethacin as well as PGE1, PGE2, PGD2, and PGI2 inhibited the proliferation of MCF-7 breast cancer cells. As breast carcinomas were described to secrete mainly PGE2, we studied the effect of PGE2 on MCF-7 cells. These cells contain two types of binding sites for PGE2: high-affinity (Kd = 0.2 nM) and low-affinity (Kd = 20 nM) receptors. In this cell line, indomethacin and PGE2 inhibitory effects were additive. In addition, we showed that PGE2 increased the cAMP level in MCF-7 cells 30-fold (p < 0.001) while indomethacin did not change basal cAMP accumulation. Like for combination PGE2/indomethacin, the inhibitory effects of a cAMP analog (8-Br-cAMP) and indomethacin were additive. In conclusion, indomethacin inhibits the MCF-7 growth in specific manner independently of PG synthesis, PG action and cAMP accumulation.

Long-term posttetantic changes in the reaction of neighboring neurons in microsegments of the cat motor cortex.

The long-term postsynaptic changes of mono- and polysynaptic reactions of neighboring neurons of the MC were investigated following conditioning tetanization of different afferent inputs. Modifiable synapses were found both in the cells investigated and in local neuronal circuits which included the cells, i.e., possibly in interneurons. Alternating and concurrent conditioning of thalamocortical and corticocortical input showed that, depending upon the modality of the conditioned input, the tetanization parameters, the character of the distribution of the afferents, as well as on the character of local circuits which include excitatory and inhibitory interneurons, the effectiveness of synaptic inputs to neighboring neurons varies diversely, as a result of which a specific pattern of interneuronal connections forms in a microsegment of cortex, a pattern which may be maintained over the course of tens of minutes. It was found that modifiable synapses of different types may function simultaneously in one and the same micronetwork. The investigation may be of interest in developing models of memory and learning.

Neurobiology of the integrative activity of the brain: some properties of long-term posttetanic heterosynaptic depression in the motor cortex of the cat.

It has been demonstrated that long-term posttetanic heterosynaptic depression (LTHD), manifested in the form of a prolonged decrease in the probability of monosynaptic responses of the cell to stimulation of that afferent pathway which was not activated during conditioning tetanization of another input, takes place in the neocortex, as it does in the hippocampus. LTHD is characterized by such properties as its long-term character, cooperativity, and nonspecificity of input. LTHD in the nonconditioned input and long-term posttetanic potentiation or long-term posttetanic homosynaptic depression in the conditioned input may develop both in parallel or independantly of one another. It is hypothesized on the basis of the results obtained that LTHD (as is the case with LTP and LTD) is a calcium-dependant phenomenon, and that the achievement of a specific level of depolarization of the membrane in the region of the disposition of the inactive synapses is required for its occurrence. "Contrasting," i.e., a relative increase in the efficiency of transmission in the activating synapse, may be effected through LTHD; LTHD may be one of the mechanisms underlying forgetting.

Proliferative responses of epithelial cells to 8-bromo-cyclic AMP and to a phorbol ester change during breast pathogenesis.

We have explored the relationship of changes in proliferative responses of human mammary epithelial cells to a phorbol ester (TPA) and to 8-Br-cAMP, which modulate the activities of protein kinases A and C (PKA and PKC), with breast tumour progression. Treatment with TPA had no effect on nontumorigenic cell lines established from human fibrocystic biopsies and apparently normal tissue around a tumour. In contrast, TPA strongly inhibited the proliferation of numerous human tumorigenic breast cell lines. Treatment with 8-Br-cAMP decreased the proliferation of all studied nontumorigenic and tumorigenic cell lines. We have also studied the effect of TPA and 8-Br-cAMP on growth of epithelial cells in short-term culture obtained from surgical human mammary biopsies with different states of breast disease. Both drugs enhanced growth of normal breast cells but had no significant effects on cells from biopsies with benign breast disease. In contrast, all examined cultures from breast cancer biopsies were strongly inhibited by 8-Br-cAMP. Otherwise, TPA had an inhibitory effect only in the case of invasive ductal carcinoma of grade III. Malignant Ha-ras-transformation of nontumorigenic TPA-insensitive breast HBL-100 cells induced an inhibitory effect of TPA. In addition, a TPA-insensitive MCF7 clone was much less tumorigenic in athymic mice than the parental strain shown to be inhibited by TPA. These data suggest that the two intracellular transduction pathways change at different stages of breast pathogenesis. Alterations in the PKA pathway are early events and are probably important to cell immortalization but do not necessarily lead to malignant development. In contrast, changes in PKC pathway are rather later events associated with advanced malignant transformation.

Evidence for a growth effect of epidermal growth factor on MDA-MB-231 breast cancer cells.

MDA-MB-231 is a breast epithelial cell line which possesses large amounts of epidermal growth factor (EGF) receptor on its cell surface but does not respond to EGF under standard culture conditions. 8-bromo-cyclic AMP (8Br-cAMP) and cholera-toxin treatments inhibit its growth by increasing its intracellular cAMP level. However, when inhibited in this way, MDA-MB-231 remains unresponsive to EGF. Similar effects--cAMP accumulation and inhibition of cell growth--are produced by forskolin. In addition, this substance specifically blocks MDA-MB-231 cells in G1 phase of the cell cycle. EGF is able to reverse the effect of forskolin on cell proliferation and prevents accumulation of cells in G1 phase without any change of cAMP level. Thus, only when inhibiting cell growth with forskolin does a mitogenic effect of EGF become evident. As cAMP is increased to a similar degree by all three compounds, yet only the effect of forskolin is antagonised by EGF, we suggest that a non-cAMP-mediated effect of forskolin must be considered to explain this effect. In contrast, the mitogenic effect of EGF on the NPM14T4/9 breast epithelial cell line does not change in the presence of forskolin.

Characterization of somatostatin receptors and growth inhibition by the somatostatin analogue BIM23014 in small cell lung carcinoma xenograft: SCLC-6.

Human small cell lung cancer (SCLC) is a neuroendocrine tumour with a very poor prognostic. Receptors for somatostatin-14 and its synthetic analogue BIM23014 (Lanreotide) were characterized in 3 human SCLC xenografts (SCLC-6, SCLC-10 and SCLC-75) transplanted in nude mice. The binding activity of both iodinated ligands was tested by cross-linking assay. One major complex of 57kDa was identified by both ligands in all 3 tumours. Two other minor complexes were only detected by the natural ligand: 90kDa in all 3 tumours and 70kDa in 2 out of the 3 tumours (SCLC-6 and SCLC-75). Analysed by Northern hybridization, the expression of the gene encoding for the receptor subtype I was detected in all 3 tumours whereas the expression of the receptor subtype II was only detected in 2 out of the 3 tumours (SCLC-6 and SCLC-75). No receptor subtype III transcript was observed. The relative quantification of the detected messengers and of the cross-linked complexes determined by densitometry suggested that SCLC-6 contained a large amount of somatostatin receptors. SCLC-6 growing in nude mice was used to evaluate the antiproliferative effect of BIM23014. BIM23014 (250 micrograms, b.i.d. for 5 days) significantly inhibited tumor growth and had an additive effect with cis-platinum (1.5 mg/kg/day for 2 days) when given concomitantly. Values of the relative tumour volume as compared to control were: BIM23014 alone 57%, cis-platinum alone 57% and BIM23014 + cis-platinum: 78%. These experimental data suggest that BIM23014 given alone or in combination with cis-platinum could have a therapeutic potential in the treatment of somatostatin receptor positive SCLCs.

[Long-term posttetanic changes in the reaction of adjacent neurons in microareas of the cat motor cortex]. / Dlitel'nye posttetanicheskie izmeneniia reaktsii sosednykh neironov v mikrouchastkakh motoronoi kory koshki.

The long-term posttetanic changes of mono- and polysynaptic reactions of the neighbouring neurons in the microareas of the motor cortex were studied before and after conditioning tetanization of different afferent inputs to these neurons. Consecutive or conjoint conditioning of the thalamo-cortical and the cortico-cortical inputs could induce different changes in the efficiency of the synaptic inputs to the neighbouring neurons the effect depending on the modality of the conditioned input, the parameters of tetanization, the pattern of distribution of the afferent terminals on the dendritic tree and the character of the local circuits formed by the neurons under study and connected with them excitatory and inhibitory interneurons. As a result of such changes the new patterns of interneuronal connections could appear and be stable for tens of minutes. It was shown that in the same neuronal network the different types of modifiable synapses could function simultaneously. The results might be used for construction of models of learning and memory.

[The properties of long-term posttetanic heterosynaptic depression in the cat motor cortex]. / O nekotorykh svoistvakh dlitel'noi posttetanicheskoi geterosinapticheskoi depressii v motornoi kore koshki.

It is shown that in the neocortex (like in the hippocampus) the long-term heterosynaptic depression (LTD) may exist. The effect appears as a long-term decrease of the probability of the monosynaptic responses in the afferent input to the recorded neuron after the conditioning tetanization of the other afferent input to the same neuron. The main properties of the heterosynaptic LTD are its longevity, cooperativity and lack of input specificity. The heterosynaptic LTD in the unconditioned input and the LTP or the homosynaptic LTD in the conditioned input can develop in parallel or independently of each other. It is supposed that the heterosynaptic LTD (like the LTP and the homosynaptic LTD) is Ca-dependent phenomenon and for its induction a certain level of depolarization of the membrane under the inactive (during conditioning) synapses must be achieved. The heterosynaptic LTD may provide for the "contrasting", i.e. the relative increase of the efficiency of the activated synapses, and probably be effective in such a phenomenon as forgetting.

[Prolonged homosynaptic depression of the impulse reactions of the motor cortex neurons in the cat]. / Dlitel'naia gomosinapticheskaia depressiia impul'snykh reaktsii neironov motornoi kory koshki.

It is shown that in the motor and the visual cortices of the cat the homosynaptic long-term posttetanic depression (LTD) of monosynaptic impulse responses of the cortical neurons may be induced in the tetanized input. Homosynaptic LTD appears as a decrease of the probability of the monosynaptic discharges or an increase in the latency of the monosynaptic responses. The cortical homosynaptic depression possesses the same properties as the hippocampal LTD, namely, the longevity, input specificity, cooperativity, and associativity. The possible mechanisms of the homosynaptic LTD induction are discussed. The effect may be determined, on the one hand, as Ca-dependent phenomenon, and on the other hand, as the LTP of monosynaptic reactions of the input inhibitory interneurons. It is supposed that the homosynaptic LTD of the impulse reactions of the cortical neurons may be one of the basic mechanisms in certain learning tasks, such as habituation or extinction.

Molecular heterogeneity of somatostatin analogue BIM-23014C receptors in human breast carcinoma cells using the chemical cross-linking assay.

Distinct proteins complexed with somatostatin and the somatostatin analogue BIM-23014C were revealed in human breast cancer cells using the cross-linking assay. One BIM-23014C-specific complex (Mr 57,000) was observed in MCF-7 (monolayer, nodule, and tumor) and T47D. Growth inhibition of MCF-7 tumor xenografts by BIM-23014C was dose related in the 6-day subrenal capsule assay. Three complexes (Mr 27,000, 42,000, and 57,000) were detected in MDA-MB-231, and no complex was visible in HBL-100. No correlation was found between receptors for BIM-23014C and epidermal growth factor in these lines. Twenty-seven of 30 human breast tumors (90%) had at least one BIM-23014C receptor. Sixteen had three complexes (Mr 27,000, 42,000, and 57,000). Six had the two complexes (Mr 27,000 and 57,000), two had Mr 42,000 and 57,000 complexes, two had just the Mr 27,000 complex, and one had just the Mr 42,000 complex. The presence of the three BIM-23014C receptors was positively correlated (P less than 0.05) to the low amount of sex steroid receptors (less than 20 fmol/mg) [seven of eight (estrogen receptor negative, progesterone receptor negative) versus four of 14 (estrogen receptor positive, progesterone receptor positive)]. Another positive correlation was established between the absence of progesterone receptors and the presence of these three complexes [12 of 16 (progesterone receptor negative) versus four of 14 (progesterone receptor positive)]. This high percentage of BIM-23014C receptor-positive biopsies and its inhibitory activity would support its clinical potential for the treatment of breast cancer.

Growth of human breast cancer cell lines is inhibited by the somatostatin analog BIM23014.

Somatostatin has been shown to lower plasma levels of various hormones and growth factors involved in regulation of the growth of human breast cells. In the present study we examined the ability of the somatostatin octapeptide analog BIM23014 to modulate the in vitro growth of five human breast cell lines: HBL100, Hs578T, MDAMB231, T47D, and MCF7. BIM23014 inhibited the growth of the two steroid-dependent cell lines, MCF7 and T47D, in a dose-related manner. This inhibitory effect was only observed when MCF7 and T47D cells were cultivated in medium containing steroid-depleted serum. The growth of a MCF7 variant capable of growth in serum-free medium was also inhibited by BIM23014, indicating that serum factors are not required for this inhibition. In the serum-free medium, the addition of estradiol before or during treatment with BIM23014 abolished its inhibitory effects on cell growth. The studies including time course, competitive inhibition, and cross-linking of iodinated BIM23014 to its receptor revealed a specific binding on MCF7 cells and showed a single 57,000 mol wt protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results support the hypothesis that BIM23014 inhibits the growth of steroid-receptor positive cells of human breast cancers through its own receptor in estradiol-free conditions.