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1. INTRODUCTION
Different neuropeptides and hormones have been identified in breast cancer tissue, and breast cancer cell lines. They include growth hormone, prolactin, vasoactive intestinal polypeptide, GnRH, GHRH, IGF-1, andfinally somatostatin and opioids. These two peptides are the major negative regulators in mammals, implicated in a variety of processes, from hormone secretion to the modulation of cell proliferation. In the present review, we will focus on the role and possible interactions of these two systems in the human breast. 2. ROLE OF SOMATOSTATIN AND OPIOID SYSTEM IN THE BREAST.
2.1 Somatostatin system Somatostatin, a naturally occurring deka-tetrapeptide, is produced mainly in the hypo-thalamus and the pancreas, but it has also been identified in a variety of normal and cancer tissues. Two main forms of somatostatin are present in biological fluids: Somatostatin 14 and somatostatin 28. The biological action of somatostatin is mediated through specific receptors, belonging to the seven-loop transmembrane receptor superfamily. Five different receptors (named SSTR1-5) have been cloned and pharmacologically characterized in a variety of tissues.In normal breast tissue, somatostatin immunoreactivity was detected in the stroma only, while epithelial cells show somatostatin production only after malignant transformation. In addition somatostatin receptors have been identified in breast cancer tissue, and human breast cancer cell lines. The existence of somatostatin receptors has been
correlated in a great number of studies with other prognostic factors. The results are contradictory, depending on the number of tumors studied and the method of analysis, but, the general conclusion is that the assay of somatostatin receptors does not have any predictive significance on the development of the disease or the response to treatment. Identification of receptor subtypes has also been assayed in malignant breast tissue. SSTR2 was the most abundantly found subtype in the breast, followed by SSTR3. In other studies, and depending on the technique used, SSRT1 and SSTR4 were equally identified, but in a much lesser extend than the two other subtypes. Finally, peritumoral blood veins, seem also to possess somatostatin receptors, and this finding indicates a possible regulatory mechanism for tumor growth. The action of somatostatin in the breast might be direct or indirect. Direct action includes the inhibitory effect of the agent on cell proliferation This effect is dose dependant, occurring at concentrations similar to the affinity of somatostatin for its receptors. It was verified in a number of cell lines, both hormone-sensitive and independent, as well as in transplantable tumors. Indirect action implies the effect of somatostatin on growth hormone secretion and IGF-1 concentration. This later factor was indeed found to have a stimulatory effect on cell growth. Nevertheless, clinical trials in small or larger series, with the use of somatostatin alone or in combination with other factors do not have shown, until now any positive result. A final implication of the somatostatinergic system in diagnosis is the scintigraphy with somatostatin analogs. It has been used during the last years, and although it was reported that it could distinguish breast cancer tumors, their metastasis and finally metastatic tumors in which the primary focus was not identified, its discriminative value was not judged satisfactory, unless with the somatostatin analogs used until now, with the possible exception of intraoperative detection. 2.2 Opioid system
Endogenous opioids derive from three different precursor proteins, namely proenkephalin A and B (prodynorphin) and proopiomelanocortin (POMC). These three proteins give rise to a number of opioid peptides with different affinities towards opioid receptors. These later receptors belong equally to the seven-loop membrane receptor superfamily, and are distinguished both pharmacologically and biochemically to three main categories: delta, mu and kappa. In addition, pharmacological evidences exist about a further subdivision of delta receptors to delta 1 and 2, of mu receptors to mu1 and mu2 and finally ofkappa receptors to kappa.
In addition to endogenous opioid peptides, a number of food derives opioids have been identified, from different proteins including hemoglobin, gluten, and caseins. Both alpha and beta caseins include peptides with a potent opioid activity (see Kampa et al, for a discussion). These later have been tested and found to decrease the proliferation of breast and prostate cells. In addition, we have identified a very potent opioid pentapeptide, derived from human alphas1-casein, with remarkable antiproliferative activity in different systems, including bread, prostate, bladder (in preparation), kidney, and different cells of hemopoietic origin (in preparation). It was named alphas1-casomorphin,and its structure is Tyr-Val-Pro-Phe-Pro-NH2 .
Opioids were found in a great diversity of primary human tumors. Between them breast tumors were found to possess endogenous opioid peptides by different technique. Furthermore, opioid receptors were equally found in primary tumors, as well as in different breast cancer cell lines. Using opioid alkaloids, endogenous opioid peptide analogs and casomorphin peptides, we, as well as other groups have shown that a dose-dependent and mostly reversible inhibition of cell proliferation can be obtained. Nevertheless, either the reversibility of this effect was not complete, either opioid agonists were not inhibited by selective antagonists. In addition, as was the case for morphine, no specific receptors were identified. These results indicate a possible mediation of the antiproliferative effect by another membrane receptor system.In general seven loops membrane receptors act through adenylate cyclase increased, or decreased activity. Somatostatin and opioid receptors decrease intracellular cAMP, through an inhibition of adenylate cyclase. In addition, in breast cancer cell lines, as well as in different other organs (kidney, prostate), we have identified further mechanisms of opioid action: Interaction with the cellular cytoskeletal element, both after an acute and a prolonged application. Furthermore, a decrease of steroid receptor levels, and a decrease of the steroid regulated secretion of enzymes were also identified. These different mechanisms, dependent and/or independent from steroid hormones and cAMP, indicate that the action of these drugs might be much more extended from previously described. It is further interesting to note that αS1-casomorphin is a very potent substance for all of the above actions, indicating that, probably, this peptide, might be a promising agent in cancer chemotherapy.
3. INTERACTION BETWEEN OPIOIDAND SOMATOSTATIN SYSTEMS The seven loop membrane receptor superfamily possesses a number of homologies between its members (see Kieffer et al, for a description). This result, in addition with the possible coexpression of multiple receptors on the somatostatin and the EGF, VIP, or the opioid receptor, and the opioid with the adrenergic, or the dopamine receptor.The coexpression of opioids and somatostatin receptors in breast cancer cell lines, the inhibitory role of both systems in cell proliferation, and a possible common mode of action of these two families of receptors, via an inhibition of adenylate cyclase together with the partial inhibition of opioid agonists by selective antagonists, makes possible such an interaction. We have indeed reported that some opioid alkaloids inhibit somatostatin binding in the human T47D breast cancer cell line. In addition, different casomorphins interact equally with somatostatin receptor, indicating that this cross talking, at the receptor level might be of a biological importance. This interaction is not restricted in the breast, but was also found in other organs as the prostate and the kidney Therefore, this effect might be a general possible cross-talking between the two main inhibitory mammalian systems. Finally, mathematical simulations have shown that, although several systems could share a common signalling pathway, they might have compartmentated intracellular access to second messengers, such as cyclase or GTPasas molecules.
4. CONCLUSION In view of the above results, a possible scheme emerges for the role of opioids and somatostatin in breast cancer. It is presented in Fig.1. In this hypothetical action, cancer cells become depolarized by their malignant transformation. In addition they produce a disruption of the basal membrane. Therefore, their secretion, which normally occurs towards the acinar lumen occurs equally towards the stroma. Stromal cells, and transformed epithelial cancer cells, produce high amounts of different proteases, which in turn could attack and partially degrade casein molecules, giving rise to casomorphin peptides. These peptides could bind to opioid receptors of malignant cells, inhibiting their proliferation, by the different mechanisms involved above, and by lowering the availability of the cells to steroids, which are responsible, at an early stage of carcinogenesis for growth. In addition, opioids, by binding to somatostatin receptors of the same malignant cells, and peritumoral vessels, they potentiate their inhibitory action. This scheme of biological action provides a possible control of tumor progression and spread, and could be of value at early stages of breast cancer.In addition to their production by casein degradation, opioids and somatostatin could arrive to tumor cell environment by several other sources. Indeed, the general circulation has a somatostatin and opioid content, provided by the secretion of these neuropeptides by other endocrine cells such as the hypothalamus, the pituitary, the pancreas or the adrenals. Local production, especially by neuroendocrine cells, production by infiltrating lymphocytes, is another potential source of these neuropeptides. It is therefore plausible to propose that these two systems (opioids and somatostatin) may play a crucial role for the development of tumor control, especially at initial stages, and that possible pharmaceutical substances, acting via these two types of receptors might be a valuable addition for cancer control.
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Somatostatin-Receptors